53010, a human carboxylesterase family member and uses thereof

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 53010 nucleic acid molecules, which encode novel carboxylesterase members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 53010 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 53010 gene has been introduced or disrupted. The invention still further provides isolated 53010 proteins, fusion proteins, antigenic peptides and anti-53010 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.60/256,369, filed on Dec. 18, 2000, and U.S. provisional application No.60/279,508, filed on Mar. 28, 2001, the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Higher eukaryotes have many distinct esterases. Among the differenttypes of esterases are those that act on carboxylic esters, also knownas carboxylesterases. Carboxylesterases have been classified into threecategories (A, B and C) on the basis of differential patters ofinhibition by organophosphates (Myers et al. (1988) Mol. Biol. Evol.5(2):113-119). Sequence analysis of a number of type-B carboxylesterasedemonstrates their evolutionary interrelatedness. Members of the type Bcarboxylesterase family include acetylcholincarboxylesterases, mammaliancholincarboxylesterases, thyroglobulin, neuroligins, and mammalian bilesalt activated lipases.

The type B family of carboxylesterase also includes vitamin K-dependentcarboxylases. Vitamin K-dependent gamma-glutamyl carboxylases catalyzethe posttranslational conversion of glutamic acid togamma-carboxyglutamic acid, an amino acid critical to the function ofthe vitamin K-dependent blood coagulation proteins (Begley et al. (2000)J. Biol. Chem. 275:36245-36249). Incomplete gamma carboxylation of bloodclotting factors is associated with poor coagulation.

For the gamma carboxylation event to occur, both vitamin K and thepresence of a gamma carboxylation recognition site on the substrate arerequired. Gamma carboxyglutamic acid confers calcium binding abilityupon the modified protein. For blood clotting factors, calcium bindingresults in a conformational change that exposes hydrophobic residues forinteractions with membranes.

Although gamma carboxylation was a biochemical event first characterizedin the mammalian blood clotting cascade, it has been found to have amore generalized applicability. For example, vitamin K-dependent gammacarboxylation of glutamate residues has also been detected for a varietyof other proteins including bone proteins, PRGP1, PRGP2, andneuropeptides (Walker et al. (2000) J. Biol. Chem., December 7 epubahead of print).

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of a novelcarboxylesterase family member, referred to herein as “53010.” Thenucleotide sequence of a cDNA encoding 53010 is shown in SEQ ID NO:1,and the amino acid sequence of a 53010 polypeptide is shown in SEQ IDNO:2. In addition, the nucleotide sequences of the coding region aredepicted in SEQ ID NO:3.

Accordingly, in one aspect, the invention features a nucleic acidmolecule that encodes a 53010 protein or polypeptide, e.g., abiologically active portion of the 53010 protein. In a preferredembodiment the isolated nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO:2. In other embodiments, theinvention provides isolated 53010 nucleic acid molecules having thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, a full complementof SEQ ID NO:1 or SEQ ID NO:3. In still other embodiments, the inventionprovides nucleic acid molecules that are substantially identical (e.g.,naturally occurring allelic variants) to the nucleotide sequence shownin SEQ ID NO:1, SEQ ID NO:3. In other embodiments, the inventionprovides a nucleic acid molecule which hybridizes under a stringencycondition described herein to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, wherein the nucleicacid encodes a full length 53010 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs that include a 53010 nucleic acid molecule described herein.In certain embodiments, the nucleic acid molecules of the invention areoperatively linked to native or heterologous regulatory sequences. Alsoincluded, are vectors and host cells containing the 53010 nucleic acidmolecules of the invention e.g., vectors and host cells suitable forproducing 53010 nucleic acid molecules and polypeptides.

In another related aspect, the invention provides nucleic acid fragmentssuitable as primers or hybridization probes for the detection of53010-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules thatare antisense to a 53010 encoding nucleic acid molecule are provided.

In another aspect, the invention features, 53010 polypeptides, andbiologically active or antigenic fragments thereof that are useful,e.g., as reagents or targets in assays applicable to treatment anddiagnosis of 53010-mediated or -related disorders. In anotherembodiment, the invention provides 53010 polypeptides having a 53010activity. Preferred polypeptides are 53010 proteins including at leastone carboxylesterase domain, and, preferably, having a 53010 activity,e.g., a 53010 activity as described herein.

In other embodiments, the invention provides 53010 polypeptides, e.g., a53010 polypeptide having the amino acid sequence shown in SEQ ID NO:2or; an amino acid sequence that is substantially identical to the aminoacid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded bya nucleic acid molecule having a nucleotide sequence which hybridizesunder a stringency condition described herein to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, whereinthe nucleic acid encodes a full length 53010 protein or an activefragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 53010 nucleic acid molecule described herein.

In a related aspect, the invention provides 53010 polypeptides orfragments operatively linked to non-53010 polypeptides to form fusionproteins.

In another aspect, the invention features antibodies and antigen-bindingfragments thereof, that react with, or more preferably specifically bind53010 polypeptides or fragments thereof, e.g., a carboxylesterasedomain.

In another aspect, the invention provides methods of screening foragents, e.g., compounds, that modulate the expression or activity of the53010 polypeptides or nucleic acids, e.g., compounds that modulate thenormal pain response, aberrant or altered pain response, or neurologicalresponse.

In a preferred embodiment, the effect of an agent, e.g., a compound, onthe pain response is evaluated by an analgesic test, e.g., the hot platetest, tail flick test, writhing test, paw pressure test, all electricstimulation test, tail withdrawal test, or formalin test.

In a preferred embodiment, the agent, e.g., compound, inhibits 53010activity.

In still another aspect, the invention provides a process for modulating53010 polypeptide or nucleic acid expression or activity, e.g. using thescreened compounds. In certain embodiments, the methods involvetreatment of conditions related to aberrant, e.g., decreased orincreased expression of the 53010 polypeptides or nucleic acids, such asconditions involving pain response, aberrant or altered pain response,or pain related disorders.

In still another aspect, the invention features a method of modulating(e.g., enhancing or inhibiting) an activity of a cell (e.g., a neuralcell), or a response (e.g., a pain response) in a subject. The methodincludes contacting the cell with, or administered to the subject, anagent, e.g., a compound, that modulates the activity or expression of a53010 polypeptide or nucleic acid, in an amount effective to modulatethe activity or the response.

In a preferred embodiment, the agent modulates (e.g., increases ordecreases) carboxylesterase activity.

In a preferred embodiment, the agent modulates (e.g., increases ordecreases) expression of the 53010 nucleic acid by, e.g., modulatingtranscription, mRNA stability, etc.

In a preferred embodiment, the cell, e.g., the 53010-expressing cell, isa central or peripheral nervous system cell, e.g., a cell in an areainvolved in pain control, e.g., a cell in the brain or spinal cord.

In a preferred embodiment, the agent, e.g., the compound, and the53010-polypeptide or nucleic acid are contacted in vitro or ex vivo. Ina preferred embodiment, the contacting step is effected in vivo in asubject, e.g., as part of a therapeutic or prophylactic protocol. Thecontacting or administering step(s) can be repeated.

Preferably, the subject is a human, e.g., a patient with pain or apain-associated disorder disclosed herein. For example, the subject canbe a patient with pain elicited from tissue injury, e.g., inflammation,infection, ischemia; pain associated with musculoskeletal disorders,e.g., joint pain; tooth pain; headaches, e.g., migrane; pain associatedwith surgery; pain related to inflammation, e.g., irritable bowelsyndrome; or chest pain. The subject can be a patient with complexregional pain syndrome (CRPS), reflex sympathetic dystrophy (RSD),causalgia, neuralgia, central pain and dysesthesia syndrome,carotidynia, neurogenic pain, refractory cervicobrachial pain syndrome,myofascial pain syndrome, craniomandibular pain dysfunction syndrome,chronic idiopathic pain syndrome, Costen's pain-dysfunction, acute chestpain syndrome, gynecologic pain syndrome, patellofemoral pain syndrome,anterior knee pain syndrome, recurrent abdominal pain in children,colic, low back pain syndrome, neuropathic pain, phantom pain fromamputation, phantom tooth pain, or pain asymbolia. The subject can be acancer patient, e.g., a patient with brain cancer, bone cancer, orprostate cancer. In other embodiments, the subject is a non-humananimal, e.g., an experimental animal, e.g., an arthritic rat model ofchronic pain, a chronic constriction injury (CCI) rat model ofneuropathic pain, or a rat model of unilateral inflammatory pain byintraplantar injection of Freund's complete adjuvant (FCA).

In other embodiments, the subject is a human, e.g., a patient withinfertility. The subject can be a cancer patient, e.g., a patient withprostate cancer. In yet other embodiments, the subject is a non-humananimal, e.g., an experimental animal, e.g., a rodent model forinfertility.

In preferred embodiments, the agent is a peptide, a phosphopeptide, asmall molecule, e.g., a member of a combinatorial library, or anantibody, or any combination thereof. The antibody can be conjugated toa therapeutic moiety selected from the group consisting of a cytotoxin,a cytotoxic agent and a radioactive metal ion.

In additional preferred embodiments, the agent is an antisense molecule,a ribozyme, a triple helix molecule, or a 53010 nucleic acid, or anycombination thereof.

In a preferred embodiment, the agent is administered in combination witha cytotoxic agent.

In another aspect, the invention features a method of treating orpreventing, in a subject, a 53010-associated disorder. The methodincludes administering to the subject, e.g., a subject at risk of, orafflicted with, a 53010-associated disorder, an agent, e.g., a compoundas described herein, that modulates the activity or expression of a53010 polypeptide or nucleic acid, in an amount effective to treat orprevent the disorder.

In a preferred embodiment, the disorder is pain or a pain relateddisorder.

In a preferred embodiment, the subject is a subject as described herein,e.g., a human.

In still another aspect, the invention features a method for evaluatingthe efficacy of a treatment of a disorder, e.g., a disorder disclosedherein, in a subject. The method includes treating a subject with aprotocol under evaluation; assessing the expression of a 53010 nucleicacid or 53010 polypeptide, such that a change in the level of 53010nucleic acid or 53010 polypeptide after treatment, relative to the levelbefore treatment, is indicative of the efficacy of the treatment of thedisorder.

In a preferred embodiment, the disorder is pain or a pain relateddisorder.

In a preferred embodiment, the subject is a human.

The invention also features a method of diagnosing a disorder, e.g., adisorder disclosed herein, in a subject. The method includes evaluatingthe expression or activity of a 53010 nucleic acid or a 53010polypeptide, such that, a difference in the level of 53010 nucleic acidor 53010 polypeptide relative to a normal subject or a cohort of normalsubjects is indicative of the disorder.

In a preferred embodiment, the disorder is pain or a pain relateddisorder.

In a preferred embodiment, the subject is a human.

In a preferred embodiment, the evaluating step occurs in vitro or exvivo. For example, a sample, e.g., a blood sample, is obtained from thesubject.

In a preferred embodiment, the evaluating step occurs in vivo. Forexample, by administering to the subject a detectably labeled agent thatinteracts with the 53010 nucleic acid or polypeptide, such that a signalis generated relative to the level of activity or expression of the53010 nucleic acid or polypeptide.

The invention also provides assays for determining the activity of orthe presence or absence of 53010 polypeptides or nucleic acid moleculesin a biological sample, including for disease diagnosis.

In further aspect, the invention provides assays for determining thepresence or absence of a genetic alteration in a 53010 polypeptide ornucleic acid molecule, including for disease diagnosis.

In yet another aspect, the invention features a method for identifyingan agent, e.g., a compound, which modulates the activity of a 53010polypeptide, e.g., a 53010 polypeptide as described herein, or theexpression of a 53010 nucleic acid, e.g., a 53010 nucleic acid asdescribed herein, including contacting the 53010 polypeptide or nucleicacid with a test agent (e.g., a test compound); and determining theeffect of the test compound on the activity of the 53010 polypeptide ornucleic acid to thereby identify a compound which modulates the activityof the 53010 polypeptide or nucleic acid.

In a preferred embodiment, the activity of the 53010 polypeptide is acarboxylesterase activity.

In a preferred embodiment, the activity of the 53010 polypeptide ismodulation of pain response.

In preferred embodiments, the agent is a peptide, a phosphopeptide, asmall molecule, e.g., a member of a combinatorial library, or anantibody, or any combination thereof.

In additional preferred embodiments, the agent is an antisense, aribozyme, or a triple helix molecule, or a 53010 nucleic acid, or anycombination thereof.

In another aspect, the invention features a two dimensional array havinga plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence. At least one address of the pluralityhas a capture probe that recognizes a 53010 molecule. In one embodiment,the capture probe is a nucleic acid, e.g., a probe complementary to a53010 nucleic acid sequence. In another embodiment, the capture probe isa polypeptide, e.g., an antibody specific for 53010 polypeptides. Alsofeatured is a method of analyzing a sample by contacting the sample tothe aforementioned array and detecting binding of the sample to thearray.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a hydropathy plot of human 53010. Relative hydrophobicresidues are shown above the dashed horizontal line, and relativehydrophilic residues are below the dashed horizontal line. Numberscorresponding to positions in the amino acid sequence of human 53010 areindicated. Polypeptides of the invention include fragments whichinclude: all or part of a hydrophobic sequence, i.e., a sequence abovethe dashed line, e.g., the sequence from about amino acid 51 to 80, fromabout 140 to 152, from about 232 to 248, and from about 410 to 435 ofSEQ ID NO:2; all or part of a hydrophilic sequence, i.e., a sequencebelow the dashed line, e.g., the sequence of from about amino acid 190to 205, from about 265 to 281, and from about 440 to 458 of SEQ ID NO:2.

FIGS. 2A-2B depict an alignment of the carboxylesterase domain of human53010 with a consensus amino acid sequence derived from a hidden Markovmodel (HMM) from PFAM. The upper sequence is the consensus amino acidsequence (SEQ ID NO:4), while the lower amino acid sequence correspondsto amino acids 44 to 545 of SEQ ID NO:2.

DETAILED DESCRIPTION

The human 53010 sequence (see SEQ ID NO:1, as recited in Example 1),which is approximately 2158 nucleotides long including untranslatedregions, contains a predicted methionine-initiated coding sequence ofabout 1746 nucleotides, including the termination codon. The codingsequence encodes a 581 amino acid protein (see SEQ ID NO:2, as recitedin Example 1). The human 53010 protein of SEQ ID NO:2 includes anamino-terminal hydrophobic amino acid sequence, consistent with a signalsequence, of about 26 amino acids (from amino acid 1 to about amino acid26 of SEQ ID NO:2), which upon cleavage results in the production of amature protein form. The mature protein form is approximately 556 aminoacid residues in length (from about amino acid 27 to amino acid 582 ofSEQ ID NO:2).

Human 53010 contains the following regions or other structural features:

a carboxylesterase domain (PFAM Accession Number PF00135) located atabout amino acid residues 44 to 545 of SEQ ID NO:2;

four predicted N-glycosylation sites (PS00001) from about amino acids287-290, 369-372, 519-522, and 530-533 of SEQ ID NO:2;

six predicted Protein Kinase C phosphorylation sites (PS00005) at aboutamino acids 75-77, 310-312, 334-336, 368-370, 425-427, and 491-493 ofSEQ ID NO:2;

eight predicted Casein Kinase II phosphorylation sites (PS00006) locatedat about amino acids 85-88, 161-164, 190-193, 289-292, 401-404, 408-411,484-487, and 554-557 of SEQ ID NO:2;

eight predicted N-myristylation sites (PS00008) from about amino acids4-9, 65-70, 153-158, 165-170, 220-225, 285-290, 367-372, and 505-510 ofSEQ ID NO:2;

a predicted ATP/GTP-binding site motif A (P-loop) (PS00017) from aboutamino acids 23-30 of SEQ ID NO:2;

a predicted carboxylesterase type-B serine active site (PS00122) fromabout amino acids 219-234 of SEQ ID NO:2; and

a predicted carboxylesterase type-B signature 2 (PS00941) from aboutamino acids 125-135 of SEQ ID NO:2.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420 andhttp://www.psc.edu/general/software/packages/pfam/pfam.html.

The 53010 protein contains a significant number of structuralcharacteristics in common with members of the carboxylesterase family.The term “family” when referring to the protein and nucleic acidmolecules of the invention means two or more proteins or nucleic acidmolecules having a common structural domain or motif and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally or non-naturally occurring and canbe from either the same or different species. For example, a family cancontain a first protein of human origin as well as other distinctproteins of human origin, or alternatively, can contain homologues ofnon-human origin, e.g., rat or mouse proteins. Members of a family canalso have common functional characteristics.

Carboxylesterase family members are esterases that act on carboxylicesters. Based on the differential patterns of inhibition byorganophosphates, carboxylesterases have been classified into threecategories (A, B and C) (Myers, M. et al. (1988) Mol. Biol. Evol.5(2):113-119). Carboxylesterase family members are characterized by acatalytic triad of amino acids: a serine, a glutamate or aspartate and ahistidine. The 53010 polypeptides described herein bear homology to thecarboxtlesterase type B family. The sequence around the active site ofcarboxtlesterase type B family members is well conserved and can be usedto form the signature patternF-[GR]-G-x(4)-[LIVM]-x-[LIV]-x-G-x-S-[STAG]-G (SEQ ID NO:5). In thispattern, S is the active site residue. A second signature pattern ofcarboxtlesterase type B family members is[ED]-D-C-L-[YT]-[LIV]-[DNS]-[LIV]-[LIVFYW]-x-[PQR] (SEQ ID NO:6), whereC is involved in a disulfide bond.

A 53010 polypeptide can include a “carboxylesterase domain” or regionshomologous with a “carboxylesterase domain”.

As used herein, the term “carboxylesterase domain” includes an aminoacid sequence of about 300 to 650 amino acid residues in length andhaving a bit score for the alignment of the sequence to thecarboxylesterase domain (HMM) of at least 300. Preferably, acarboxylesterase domain includes at least about 400-600 amino acids,more preferably about 450-550 amino acid residues, or about 490-510amino acids and has a bit score for the alignment of the sequence to thecarboxylesterase domain (HMM) of at least 400, 450, 500, 540 or greater.The carboxylesterase domain (HMM) has been assigned the PFAM AccessionNumber PF00135 (http;//genome.wustl.edu/Pfam/.html). An alignment of thecarboxylesterase domain (amino acids 44 to 545 of SEQ ID NO:2) of human53010 with a consensus amino acid sequence (SEQ ID NO:4) derived from ahidden Markov model is depicted in FIG. 2.

In a preferred embodiment 53010 polypeptide or protein has a“carboxylesterase domain” or a region which includes at least about400-600, more preferably about 450-550 or 490-510 amino acid residuesand has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homologywith a “carboxylesterase domain,” e.g., the carboxylesterase domain ofhuman 53010 (e.g., residues 44 to 545 of SEQ ID NO:2).

To identify the presence of a “carboxylesterase” domain in a 53010protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the Pfam database of HMMs (e.g., thePfam database, release 2.1) using the default parameters(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al.(1990)Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci.USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; andStultz et al.(1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference. A search was performed against the HMMdatabase resulting in the identification of a “carboxylesterase” domainin the amino acid sequence of human 53010 at about residues 44 to 545 ofSEQ ID NO:2 (see FIG. 2).

A 53010 molecule can include a carboxylesterase type-B serine activesite. As used herein, the term “carboxylesterase type-B serine activesite” includes an amino acid sequence of about 5-30, more preferably10-20, most preferably 16 amino acids in length, which includes theconsensus sequence F-[GR]-G-x(4)-[LIVM]-x-[LIV]-x-G-x-S-[STAG]-G (SEQ IDNO:5). Most preferably, the carboxylesterase type-B serine active sitehas a sequence at least 90%, 95%, 98%, or 100% identical to the aminoacid sequence FGGDPSSVTIFGESAG (located at amino acids 219-234 of SEQ IDNO:2).

A 53010 molecule can include a carboxylesterase type-B signature 2. Asused herein, the term “carboxylesterase type-B signature 2” includes anamino acid sequence of about 5-20, more preferably 8-15, most preferably11 amino acids in length, which includes the consensus sequence[ED]-D-C-L-[YT]-[LIV]-[DNS]-[LIV]-[LIVFYW]-x-[PQR] (SEQ ID NO:6). Mostpreferably, the carboxylesterase type-B signature 2 has a sequence atleast 90%, 95%, 98%, or 100% identical to the amino acid sequenceEDCLYLNIYAP (located at amino acids 125-135 of SEQ ID NO:2).

A 53010 family member can include a carboxylesterase domain; acarboxylesterase type-B serine active site; and a carboxylesterasetype-B signature 2.

Furthermore, a 53010 family member can include at least one, two, three,and preferably four N-glycosylation sites (PS00001); at least one, two,three, four, five, and preferably six protein kinase C phosphorylationsites (PS00005); at least one, two, three, four, five, six, seven, andpreferably eight predicted casein kinase II phosphorylation sites(PS00006); at least one, two, three, four, five, six, seven, andpreferably eight predicted N-myristylation sites (PS00008); and at leastone ATP/GTP-binding site motif A (PS00017);

As the 53010 polypeptides of the invention may modulate 53010-mediatedactivities, they may be useful as of for developing novel diagnostic andtherapeutic agents for 53010-mediated or related disorders, as describedbelow.

As used herein, a “53010 activity”, “biological activity of 53010” or“functional activity of 53010”, refers to an activity exerted by a 53010protein, polypeptide or nucleic acid molecule. For example, a 53010activity can be an activity exerted by 53010 in a physiological milieuon, e.g., a 53010-responsive cell or on a 53010 substrate, e.g., aprotein substrate. A 53010 activity can be determined in vivo or invitro. In one embodiment, a 53010 activity is a direct activity, such asan association with a 53010 target molecule. A “target molecule” or“binding partner” is a molecule with which a 53010 protein binds orinteracts in nature. In an exemplary embodiment, 53010 is an enzyme thatcatalyzes the hydrolysis of carboxylic esters.

A 53010 activity can also be an indirect activity, e.g., a cellularsignaling activity mediated by interaction of the 53010 protein with a53010 receptor. The features of the 53010 molecules of the presentinvention can provide similar biological activities as carboxylesterasefamily members. For example, the 53010 proteins of the present inventioncan have one or more of the following activities: (1) the ability tocatalyze the hydrolysis of carboxylic esters; (2) the ability tomodulate cell—cell recognition events, e.g., adhesion or attachment; (3)the ability to interact with a cell surface protein, an extracellularprotein, or an extracellular component; (4) the ability catalyze theposttranslational conversion of glutamic acid to gamma-carboxyglutamicacid; (5) the ability to modulate blood coagulation; (6) the ability tomodulate cell migration; (7) the ability to modulate proliferationand/or differentiation of a cell (e.g., a neural or a cancer cell); (8)the ability to modulate embryonic development and differentiation; (9)the ability to modulate tissue maintenance; (10) the ability to modulateneural development; (11) the ability to bind a divalent cation, e.g.,Zn²⁺, Mg²⁺, Cd²⁺, Mn²⁺ and/or preferably a Ca2⁺ ion; or (12) the abilityto modulate pain or inflammation response.

Thus, the 53010 molecules can act as novel diagnostic targets andtherapeutic agents for controlling disorders involving aberrant ordeficient hydrolysis of carboxylic esters.

53010 is highly expressed in the central and peripheral nervous system.For example, Tables 1-3 show that 53010 mRNA is expressed at highlevels, relative to other tissues tested, in the brain, spinal cord, anddorsal root ganglion. The rat orthologue of 53010 was found to beexpressed at low levels only in the nervous system tissues. In situhybridization experiments with a human 53010 probe showed expression of53010 in a subpopulation of neurons in the human thalamus and low levelsof expression in monkey hippocampus (CA layers), spinal cord, and dorsalroot ganglion. Low levels of 53010 expression were also detected inhuman cortex, and higher levels were observed in subpopulation ofthalamic nuclei. In rat, in situ hybridization experiments with a rat53010 probe detected expression in the brain restricted to the ventroposterior, ventrolateral and postero ventral nuclei of the thalamus.These are the nuclei that received information from the spinal cord andthe dorsal column nuclei. Low levels of expression were observed in thespinal cord. The rodent 53010 gene was found to be expressed at lowlevels in the dorsal root ganglion, except in a very few small diameterneurons that express much higher levels.

Animal models of pain response include, but are not limited to: axotomy,the cutting or severing of an axon (Gustafsson et al. (2000) Neuroreport11:3345-48); chronic constriction injury (CCI), also known as theBennett model, a model of neuropathic pain which involves ligation ofthe sciatic nerve in rodents, e.g., rats (Eaton et al. (2000) CellTransplant. 9:637-56); and intraplantar complete Freund's adjuvant (CFA)injection as a model of arthritic pain (Fraser et al. (2000) Br. J.Pharmacol. 129:1668-72). Other animal models of pain response aredescribed in, e.g., ILAR Journal (1999) Volume 40, Number 3 (entireissue). 53010 expression was shown to be slightly upregulated in somepain response models (see Tables 4 and 5).

Carboxylesterases are regulators of lipid metabolism and have a broadspecificity. They catalyze the reactions of acylglycerol lipases (thathydrolyze 2-AG and DAG into arachidonic acid). Carboxylesterases alsocatalyze amidase reactions (inactivating endogenous cannabinoids) andlysophospholipase reactions (the enzyme that produces lysophosphatidicacid, a pain-inducing factor that acts directly on the afferentterminal). Carboxylesterase inhibitors can increase the levels of 2-AG(an endogenous cannabinoid).

As the 53010 mRNA is expressed in the central and peripheral nervoussystem (e.g., brain, spinal cord, and dorsal root ganglion (DRG)) ofprimates, e.g., human and monkeys, and its expression is regulatedexpression in some rodent pain models, 53010 molecules can act as noveldiagnostic targets and therapeutic agents for controlling neurologicaldisorders, such as pain-related disorders.

Examples of pain conditions include, but are not limited to, painelicited during various forms of tissue injury, e.g., inflammation,infection, and ischemia; pain associated with musculoskeletal disorders,e.g., joint pain, or arthritis; tooth pain; headaches, e.g., migrane;pain associated with surgery; pain related to inflammation, e.g.,irritable bowel syndrome; chest pain; or hyperalgesia, e.g., excessivesensitivity to pain (described in, for example, Fields (1987) Pain, NewYork:McGraw-Hill). Other examples of pain disorders or pain syndromesinclude, but are not limited to, complex regional pain syndrome (CRPS),reflex sympathetic dystrophy (RSD), causalgia, neuralgia, central painand dysesthesia syndrome, carotidynia, neurogenic pain, refractorycervicobrachial pain syndrome, myofascial pain syndrome,craniomandibular pain dysfunction syndrome, chronic idiopathic painsyndrome, Costen's pain-dysfunction, acute chest pain syndrome, nonulcerdyspepsia, interstitial cystitis, gynecologic pain syndrome,patellofemoral pain syndrome, anterior knee pain syndrome, recurrentabdominal pain in children, colic, low back pain syndrome, neuropathicpain, phantom pain from amputation, phantom tooth pain, or painasymbolia (the inability to feel pain). Other examples of painconditions include pain induced by parturition, or post partum pain.

Agents that modulate 53010 polypeptide or nucleic acid activity orexpression can be used to treat pain elicited by any medical condition.A subject receiving the treatment can be additionally treated with asecond agent, e.g., an anti-inflammatory agent, an antibiotic, or achemotherapeutic agent, to further ameliorate the condition.

The 53010 molecules can also act as novel diagnostic targets andtherapeutic agents controlling pain caused by other disorders, e.g.,cancer, e.g., prostate cancer.

The molecules of the invention may also serve as diagnostic andtherapeutic targets for neurological disorders in addition to the onesdescribed above. Examples of such neurological disorders include, butare not limited to, disorders involving neurons, and disorders involvingglia, such as astrocytes, oligodendrocytes, ependymal cells, andmicroglia; cerebral edema, raised intracranial pressure and herniation,and hydrocephalus; malformations and developmental diseases, such asneural tube defects, forebrain anomalies, posterior fossa anomalies, andsyringomyelia and hydromyelia;

perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

The 53010 protein, fragments thereof, and derivatives and other variantsof the sequence in SEQ ID NO:2 thereof are collectively referred to as“polypeptides or proteins of the invention” or “53010 polypeptides orproteins”. Nucleic acid molecules encoding such polypeptides or proteinsare collectively referred to as “nucleic acids of the invention” or“53010 nucleic acids.” 53010 molecules refer to 53010 nucleic acids,polypeptides, and antibodies.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogsof the DNA or RNA. A DNA or RNA analog can be synthesized fromnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated nucleic acid molecule” or “purified nucleic acidmolecule” includes nucleic acid molecules that are separated from othernucleic acid molecules present in the natural source of the nucleicacid. For example, with regards to genomic DNA, the term “isolated”includes nucleic acid molecules which are separated from the chromosomewith which the genomic DNA is naturally associated. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2× SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6× SSC at about 45° C., followed by one or more washes in 0.2× SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6× SSC atabout 45° C., followed by one or more washes in 0.2× SSC, 0.1% SDS at65° C.; and preferably 4) very high stringency hybridization conditionsare 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or morewashes at 0.2× SSC, 1% SDS at 65° C. Very high stringency conditions (4)are the preferred conditions and the ones that should be used unlessotherwise specified.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under a stringency condition described herein to the sequenceof SEQ ID NO:1 or SEQ ID NO:3, corresponds to a naturally-occurringnucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature. For example a naturally occurring nucleic acid molecule canencode a natural protein. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include atleast an open reading frame encoding a 53010 protein. The gene canoptionally further include non-coding sequences, e.g., regulatorysequences and introns. Preferably, a gene encodes a mammalian 53010protein or derivative thereof.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.“Substantially free” means that a preparation of 53010 protein is atleast 10% pure. In a preferred embodiment, the preparation of 53010protein has less than about 30%, 20%, 10% and more preferably 5% (by dryweight), of non-53010 protein (also referred to herein as a“contaminating protein”), or of chemical precursors or non-53010chemicals. When the 53010 protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The invention includesisolated or purified preparations of at least 0.01, 0.1, 1.0, and 10milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of 53010 without abolishing or substantiallyaltering a 53010 activity. Preferably the alteration does notsubstantially alter the 53010 activity, e.g., the activity is at least20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acidresidue is a residue that, when altered from the wild-type sequence of53010, results in abolishing a 53010 activity such that less than 20% ofthe wild-type activity is present. For example, conserved amino acidresidues in 53010 are predicted to be particularly unamenable toalteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a 53010 protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a 53010 coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor 53010 biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1 or SEQ ID NO:3, the encoded proteincan be expressed recombinantly and the activity of the protein can bedetermined.

As used herein, a “biologically active portion” of a 53010 proteinincludes a fragment of a 53010 protein which participates in aninteraction, e.g., an intramolecular or an inter-molecular interaction.An inter-molecular interaction can be a specific binding interaction oran enzymatic interaction (e.g., the interaction can be transient and acovalent bond is formed or broken). An inter-molecular interaction canbe between a 53010 molecule and a non-53010 molecule or between a first53010 molecule and a second 53010 molecule (e.g., a dimerizationinteraction). Biologically active portions of a 53010 protein includepeptides comprising amino acid sequences sufficiently homologous to orderived from the amino acid sequence of the 53010 protein, e.g., theamino acid sequence shown in SEQ ID NO:2, which include less amino acidsthan the full length 53010 proteins, and exhibit at least one activityof a 53010 protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the 53010 protein, e.g.,the ability to catalyze the hydrolysis of carboxylic esters. Abiologically active portion of a 53010 protein can be a polypeptidewhich is, for example, 10, 25, 50, 100, 200 or more amino acids inlength. Biologically active portions of a 53010 protein can be used astargets for developing agents which modulate a 53010 mediated activity,e.g., the ability to catalyze the hydrolysis of carboxylic esters.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 53010 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 53010 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

Particularly preferred 53010 polypeptides of the present invention havean amino acid sequence substantially identical to the amino acidsequence of SEQ ID NO:2. In the context of an amino acid sequence, theterm “substantially identical” is used herein to refer to a first aminoacid that contains a sufficient or minimum number of amino acid residuesthat are i) identical to, or ii) conservative substitutions of alignedamino acid residues in a second amino acid sequence such that the firstand second amino acid sequences can have a common structural domainand/or common functional activity. For example, amino acid sequencesthat contain a common structural domain having at least about 60%, or65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2 are termedsubstantially identical.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 or 3 are termedsubstantially identical. “Misexpression or aberrant expression”, as usedherein, refers to a non-wildtype pattern of gene expression at the RNAor protein level. It includes: expression at non-wild type levels, i.e.,over- or under-expression; a pattern of expression that differs fromwild type in terms of the time or stage at which the gene is expressed,e.g., increased or decreased expression (as compared with wild type) ata predetermined developmental period or stage; a pattern of expressionthat differs from wild type in terms of altered, e.g., increased ordecreased, expression (as compared with wild type) in a predeterminedcell type or tissue type; a pattern of expression that differs from wildtype in terms of the splicing size, translated amino acid sequence,post-transitional modification, or biological activity of the expressedpolypeptide; a pattern of expression that differs from wild type interms of the effect of an environmental stimulus or extracellularstimulus on expression of the gene, e.g., a pattern of increased ordecreased expression (as compared with wild type) in the presence of anincrease or decrease in the strength of the stimulus.

“Subject,” as used herein, refers to human and non-human animals. Theterm “non-human animals” of the invention includes all vertebrates,e.g., mammals, such as non-human primates (particularly higherprimates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat,pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians,reptiles, etc. In a preferred embodiment, the subject is a human. Inanother embodiment, the subject is an experimental animal or animalsuitable as a disease model.

A “purified preparation of cells”, as used herein, refers to an in vitropreparation of cells. In the case cells from multicellular organisms(e.g., plants and animals), a purified preparation of cells is a subsetof cells obtained from the organism, not the entire intact organism. Inthe case of unicellular microorganisms (e.g., cultured cells andmicrobial cells), it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleicacid molecule that encodes a 53010 polypeptide described herein, e.g., afull-length 53010 protein or a fragment thereof, e.g., a biologicallyactive portion of 53010 protein. Also included is a nucleic acidfragment suitable for use as a hybridization probe, which can be used,e.g., to identify a nucleic acid molecule encoding a polypeptide of theinvention, 53010 mRNA, and fragments suitable for use as primers, e.g.,PCR primers for the amplification or mutation of nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence shown in SEQ ID NO:1, or a portion ofany of these nucleotide sequences. In one embodiment, the nucleic acidmolecule includes sequences encoding the human 53010 protein (i.e., “thecoding region” of SEQ ID NO:1, as shown in SEQ ID NO:3), as well as 5′untranslated sequences. Alternatively, the nucleic acid molecule caninclude only the coding region of SEQ ID NO:1 (e.g., SEQ ID NO:3) and,e.g., no flanking sequences which normally accompany the subjectsequence. In another embodiment, the nucleic acid molecule encodes asequence corresponding to a fragment of the protein from about aminoacid 44 to 545.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement, e.g.,a full complement, of the nucleotide sequence shown in SEQ ID NO:1 orSEQ ID NO:3, or a portion of any of these nucleotide sequences. In otherembodiments, the nucleic acid molecule of the invention is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NO:1 or SEQ IDNO:3, such that it can hybridize (e.g., under a stringency conditiondescribed herein) to the nucleotide sequence shown in SEQ ID NO:1 or 3,thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about: 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more homologous to the entire length of the nucleotide sequenceshown in SEQ ID NO:1 or SEQ ID NO:3, or a portion, preferably of thesame length, of any of these nucleotide sequences.

53010 Nucleic Acid Fragments

A nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:1 or 3. For example, such anucleic acid molecule can include a fragment which can be used as aprobe or primer or a fragment encoding a portion of a 53010 protein,e.g., an immunogenic or biologically active portion of a 53010 protein.A fragment can comprise those nucleotides of SEQ ID NO:1, which encode acarboxylesterase domain of human 53010. The nucleotide sequencedetermined from the cloning of the 53010 gene allows for the generationof probes and primers designed for use in identifying and/or cloningother 53010 family members, or fragments thereof, as well as 53010homologues, or fragments thereof, from other species.

In another embodiment, a nucleic acid includes a nucleotide sequencethat includes part, or all, of the coding region and extends into either(or both) the 5′ or 3′ noncoding region. Other embodiments include afragment which includes a nucleotide sequence encoding an amino acidfragment described herein. Nucleic acid fragments can encode a specificdomain or site described herein or fragments thereof, particularlyfragments thereof which are at least 50, 100, 150, 200, 250, 300, 350,400, 450, 500, or 550 amino acids in length. Fragments also includenucleic acid sequences corresponding to specific amino acid sequencesdescribed above or fragments thereof. Nucleic acid fragments should notto be construed as encompassing those fragments that may have beendisclosed prior to the invention.

A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, a 53010 nucleic acid fragment caninclude a sequence corresponding to a carboxylesterase domain.

53010 probes and primers are provided. Typically a probe/primer is anisolated or purified oligonucleotide. The oligonucleotide typicallyincludes a region of nucleotide sequence that hybridizes under astringency condition described herein to at least about 7, 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense or antisense sequenceof SEQ ID NO: 1 or SEQ ID NO:3, or of a naturally occurring allelicvariant or mutant of SEQ ID NO:1 or SEQ ID NO:3. Preferably, anoligonucleotide is less than about 200, 150, 120, or 100 nucleotides inlength.

In one embodiment, the probe or primer is attached to a solid support,e.g., a solid support described herein.

One exemplary kit of primers includes a forward primer that anneals tothe coding strand and a reverse primer that anneals to the non-codingstrand. The forward primer can anneal to the start codon, e.g., thenucleic acid sequence encoding amino acid residue 1 of SEQ ID NO:2. Thereverse primer can anneal to the ultimate codon, e.g., the codonimmediately before the stop codon, e.g., the codon encoding amino acidresidue 581 of SEQ ID NO:2. In a preferred embodiment, the annealingtemperatures of the forward and reverse primers differ by no more than5, 4, 3, or 2° C.

In a preferred embodiment the nucleic acid is a probe which is at least10, 12, 15, 18, 20 and less than 200, more preferably less than 100, orless than 50, nucleotides in length. It should be identical, or differby 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosedherein. If alignment is needed for this comparison the sequences shouldbe aligned for maximum homology. “Looped” out sequences from deletionsor insertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand ofa nucleic acid which encodes: a carboxylesterase domain (e.g., aminoacids 44 to 545 of SEQ ID NO:2); a carboxylesterase type-B serine activesite (e.g., amino acids 125-135 of SEQ ID NO:2); or carboxylesterasetype-B signature 2 (e.g., amino acids 125-135 of SEQ ID NO:2).

In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 53010 sequence, e.g., a domain, region, site or othersequence described herein. The primers should be at least 5, 10, or 50base pairs in length and less than 100, or less than 200, base pairs inlength. The primers should be identical, or differs by one base from asequence disclosed herein or from a naturally occurring variant. Forexample, primers suitable for amplifying all or a portion of any of thefollowing regions are provided: a carboxylesterase domain (e.g., aminoacids 44 to 545 of SEQ ID NO:2); a carboxylesterase type-B serine activesite (e.g., amino acids 125-135 of SEQ ID NO:2); or carboxylesterasetype-B signature 2 (e.g., amino acids 125-135 of SEQ ID NO:2).

A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

A nucleic acid fragment encoding a “biologically active portion of a53010 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO: 1 or 3, which encodes a polypeptidehaving a 53010 biological activity (e.g., the biological activities ofthe 53010 proteins are described herein), expressing the encoded portionof the 53010 protein (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the 53010 protein. Forexample, a nucleic acid fragment encoding a biologically active portionof 53010 includes a carboxylesterase domain, e.g., amino acid residuesabout 44 to 545 of SEQ ID NO:2. A nucleic acid fragment encoding abiologically active portion of a 53010 polypeptide, may comprise anucleotide sequence which is greater than 300 or more nucleotides inlength.

In preferred embodiments, a nucleic acid includes a nucleotide sequencewhich is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300or more nucleotides in length and hybridizes under a stringencycondition described herein to a nucleic acid molecule of SEQ ID NO:1, orSEQ ID NO:3.

53010 Nucleic Acid Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. Suchdifferences can be due to degeneracy of the genetic code (and result ina nucleic acid which encodes the same 53010 proteins as those encoded bythe nucleotide sequence disclosed herein. In another embodiment, anisolated nucleic acid molecule of the invention has a nucleotidesequence encoding a protein having an amino acid sequence which differs,by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residuesthat shown in SEQ ID NO:2. If alignment is needed for this comparisonthe sequences should be aligned for maximum homology. The encodedprotein can differ by no more than 5, 4, 3, 2, or 1 amino acid. “Looped”out sequences from deletions or insertions, or mismatches, areconsidered differences.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non-preferred, for a particular expression system. E.g.,the nucleic acid can be one in which at least one codon, at preferablyat least 10%, or 20% of the codons has been altered such that thesequence is optimized for expression in E. coli, yeast, human, insect,or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

In a preferred embodiment, the nucleic acid differs from that of SEQ IDNO: 1 or 3, e.g., as follows: by at least one but less than 10, 20, 30,or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of thenucleotides in the subject nucleic acid. The nucleic acid can differ byno more than 5, 4, 3, 2, or 1 nucleotide. If necessary for this analysisthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.

Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the nucleotidesequence shown in SEQ ID NO:2 or a fragment of this sequence. Suchnucleic acid molecules can readily be identified as being able tohybridize under a stringency condition described herein, to thenucleotide sequence shown in SEQ ID NO 2 or a fragment of the sequence.Nucleic acid molecules corresponding to orthologs, homologs, and allelicvariants of the 53010 cDNAs of the invention can further be isolated bymapping to the same chromosome or locus as the 53010 gene.

Preferred variants include those that are correlated with the ability tocatalyze the hydrolysis of carboxylic esters.

Allelic variants of 53010, e.g., human 53010, include both functionaland non-functional proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the 53010 protein within apopulation that maintain the ability to catalyze the hydrolysis ofcarboxylic esters. Functional allelic variants will typically containonly conservative substitution of one or more amino acids of SEQ IDNO:2, or substitution, deletion or insertion of non-critical residues innon-critical regions of the protein. Non-functional allelic variants arenaturally-occurring amino acid sequence variants of the 53010, e.g.,human 53010, protein within a population that do not have the ability tocatalyze the hydrolysis of carboxylic esters. Non-functional allelicvariants will typically contain a non-conservative substitution, adeletion, or insertion, or premature truncation of the amino acidsequence of SEQ ID NO:2, or a substitution, insertion, or deletion incritical residues or critical regions of the protein.

Moreover, nucleic acid molecules encoding other 53010 family membersand, thus, which have a nucleotide sequence which differs from the 53010sequences of SEQ ID NO:1 or SEQ ID NO:3 are intended to be within thescope of the invention.

Antisense Nucleic Acid Molecules, Ribozymes and Modified 53010 NucleicAcid Molecules

In another aspect, the invention features, an isolated nucleic acidmolecule which is antisense to 53010. An “antisense” nucleic acid caninclude a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. The antisense nucleic acid can be complementary to an entire53010 coding strand, or to only a portion thereof (e.g., the codingregion of human 53010 corresponding to SEQ ID NO:3). In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding 53010 (e.g., the 5′ and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of 53010 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of 53010 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of 53010 mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence of interest. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. The antisense nucleic acid also canbe produced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a 53010 protein to thereby inhibitexpression of the protein, e.g., by inhibiting transcription and/ortranslation. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) (Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. A ribozyme having specificity for a 53010-encodingnucleic acid can include one or more sequences complementary to thenucleotide sequence of a 53010 cDNA disclosed herein (i.e., SEQ ID NO:1or SEQ ID NO:3), and a sequence having known catalytic sequenceresponsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoffand Gerlach (1988) Nature 334:585-591). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a 53010-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,53010 mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostal J. W. (1993) Science 261:1411-1418.

53010 gene expression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the 53010 (e.g., the 53010promoter and/or enhancers) to form triple helical structures thatprevent transcription of the 53010 gene in target cells. See generally,Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992)Ann. N. Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14:807-15. The potential sequences that can be targeted for triple helixformation can be increased by creating a so-called “switchback” nucleicacid molecule. Switchback molecules are synthesized in an alternating5′-3′, 3′-5′ manner, such that they base pair with first one strand of aduplex and then the other, eliminating the necessity for a sizeablestretch of either purines or pyrimidines to be present on one strand ofa duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or calorimetric.

A 53010 nucleic acid molecule can be modified at the base moiety, sugarmoiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For non-limiting examplesof synthetic oligonucleotides with modifications see Toulmë (2001)Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44.Such phosphoramidite oligonucleotides can be effective antisense agents.

For example, the deoxyribose phosphate backbone of the nucleic acidmolecules can be modified to generate peptide nucleic acids (see HyrupB. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As usedherein, the terms “peptide nucleic acid” or “PNA” refers to a nucleicacid mimic, e.g., a DNA mimic, in which the deoxyribose phosphatebackbone is replaced by a pseudopeptide backbone and only the fournatural nucleobases are retained. The neutral backbone of a PNA canallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in HyrupB. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci.93: 14670-675.

PNAs of 53010 nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of 53010 nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. etal. (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W088/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer andprobe molecules having at least one region which is complementary to a53010 nucleic acid of the invention, two complementary regions onehaving a fluorophore and one a quencher such that the molecular beaconis useful for quantitating the presence of the 53010 nucleic acid of theinvention in a sample. Molecular beacon nucleic acids are described, forexample, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al.,U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

Isolated 53010 Polypeptides

In another aspect, the invention features, an isolated 53010 protein, orfragment, e.g., a biologically active portion, for use as immunogens orantigens to raise or test (or more generally to bind) anti-53010antibodies. 53010 protein can be isolated from cells or tissue sourcesusing standard protein purification techniques. 53010 protein orfragments thereof can be produced by recombinant DNA techniques orsynthesized chemically.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andpost-translational events. The polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepost-translational modifications present when expressed the polypeptideis expressed in a native cell, or in systems which result in thealteration or omission of post-translational modifications, e.g.,glycosylation or cleavage, present when expressed in a native cell.

In a preferred embodiment, a 53010 polypeptide has one or more of thefollowing characteristics:

(i) it has the ability to catalyze the hydrolysis of carboxylic esters;

(ii) it has a molecular weight, e.g., a deduced molecular weight,preferably ignoring any contribution of post translationalmodifications, amino acid composition or other physical characteristicof a 53010 polypeptide, e.g., a polypeptide of SEQ ID NO:2;

(iii) it has an overall sequence similarity of at least 60%, morepreferably at least 70, 80, 90, or 95%, with a 53010 polypeptide, e.g.,a polypeptide of SEQ ID NO:2;

(iv) it has a carboxylesterase domain which is preferably about 70%,80%, 90% or 95% with amino acid residues about 44 to 545 of SEQ ID NO:2;

(v) it has a carboxylesterase type-B serine active site which ispreferably about 70%, 80%, 90% or 95% with amino acid residues about219-234 of SEQ ID NO:2;

(vi) it has a carboxylesterase type-B signature 2 which is preferablyabout 70%, 80%, 90% or 95% with amino acid residues about 125 to 135 ofSEQ ID NO:2; or

(vii) it has at least one, preferably five, and most preferably six ofthe cysteines found in the amino acid sequence of the native protein.

In a preferred embodiment the 53010 protein, or fragment thereof,differs from the corresponding sequence in SEQ ID NO:2. In oneembodiment it differs by at least one but by less than 15, 10 or 5 aminoacid residues. In another it differs from the corresponding sequence inSEQ ID NO:2 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ IDNO:2. (If this comparison requires alignment the sequences should bealigned for maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.) The differencesare, preferably, differences or changes at a non essential residue or aconservative substitution. In a preferred embodiment the differences arenot in the carboxylesterase domain of from about amino acid 44 to 545 ofSEQ ID NO:2. In another preferred embodiment one or more differences arein the carboxylesterase domain of from about amino acid 44 to 545 of SEQID NO:2.

Other embodiments include a protein that contain one or more changes inamino acid sequence, e.g., a change in an amino acid residue which isnot essential for activity. Such 53010 proteins differ in amino acidsequence from SEQ ID NO:2, yet retain biological activity.

In one embodiment, the protein includes an amino acid sequence at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to SEQ ID NO:2.

A 53010 protein or fragment is provided which varies from the sequenceof SEQ ID NO.2 in regions defined by amino acids about 1 to 138 by atleast one but by less than 15, 10 or 5 amino acid residues in theprotein or fragment but which does not differ from SEQ ID NO.2 inregions defined by amino acids about 44 to 545. (If this comparisonrequires alignment the sequences should be aligned for maximum homology.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences.) In some embodiments the difference is at anon-essential residue or is a conservative substitution, while in othersthe difference is at an essential residue or is a non-conservativesubstitution.

In one embodiment, a biologically active portion of a 53010 proteinincludes a carboxylesterase domain. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native 53010 protein.

In a preferred embodiment, the 53010 protein has an amino acid sequenceshown in SEQ ID NO:2. In other embodiments, the 53010 protein issubstantially identical to SEQ ID NO:2. In yet another embodiment, the53010 protein is substantially identical to SEQ ID NO:2 and retains thefunctional activity of the protein of SEQ ID NO:2, as described indetail in the subsections above.

53010 Chimeric or Fusion Proteins

In another aspect, the invention provides 53010 chimeric or fusionproteins. As used herein, a 53010 “chimeric protein” or “fusion protein”includes a 53010 polypeptide linked to a non-53010 polypeptide. A“non-53010 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the 53010 protein, e.g., a protein which is different fromthe 53010 protein and which is derived from the same or a differentorganism. The 53010 polypeptide of the fusion protein can correspond toall or a portion e.g., a fragment described herein of a 53010 amino acidsequence. In a preferred embodiment, a 53010 fusion protein includes atleast one (or two) biologically active portion of a 53010 protein. Thenon-53010 polypeptide can be fused to the N-terminus or C-terminus ofthe 53010 polypeptide.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-53010 fusionprotein in which the 53010 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant 53010. Alternatively, the fusion protein can be a 53010protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 53010 can be increased through use of a heterologous signalsequence.

Fusion proteins can include all or a part of a serum protein, e.g., anIgG constant region, or human serum albumin.

The 53010 fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. The53010 fusion proteins can be used to affect the bioavailability of a53010 substrate. 53010 fusion proteins may be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 53010 protein; (ii)mis-regulation of the 53010 gene; and (iii) aberrant post-translationalmodification of a 53010 protein.

Moreover, the 53010-fusion proteins of the invention can be used asimmunogens to produce anti-53010 antibodies in a subject, to purify53010 ligands and in screening assays to identify molecules whichinhibit the interaction of 53010 with a 53010 substrate.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A 53010-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the 53010 protein.

Variants of 53010 Proteins

In another aspect, the invention also features a variant of a 53010polypeptide, e.g., which functions as an agonist (mimetics) or as anantagonist. Variants of the 53010 proteins can be generated bymutagenesis, e.g., discrete point mutation, the insertion or deletion ofsequences or the truncation of a 53010 protein. An agonist of the 53010proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a 53010protein. An antagonist of a 53010 protein can inhibit one or more of theactivities of the naturally occurring form of the 53010 protein by, forexample, competitively modulating a 53010-mediated activity of a 53010protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Preferably, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of the53010 protein.

Variants of a 53010 protein can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of a 53010 protein foragonist or antagonist activity.

Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 53010 protein coding sequence can be used to generate avariegated population of fragments for screening and subsequentselection of variants of a 53010 protein. Variants in which a cysteineresidues is added or deleted or in which a residue which is glycosylatedis added or deleted are particularly preferred.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of 53010 proteins. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify 53010 variants (Arkin and Yourvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) ProteinEngineering 6:327-331).

Cell based assays can be exploited to analyze a variegated 53010library. For example, a library of expression vectors can be transfectedinto a cell line, e.g., a cell line, which ordinarily responds to 53010in a substrate-dependent manner. The transfected cells are thencontacted with 53010 and the effect of the expression of the mutant onsignaling by the 53010 substrate can be detected, e.g., by measuringcarboxylesterase activity. Plasmid DNA can then be recovered from thecells which score for inhibition, or alternatively, potentiation ofsignaling by the 53010 substrate, and the individual clones furthercharacterized.

In another aspect, the invention features a method of making a 53010polypeptide, e.g., a peptide having a non-wild type activity, e.g., anantagonist, agonist, or super agonist of a naturally occurring 53010polypeptide, e.g., a naturally occurring 53010 polypeptide. The methodincludes: altering the sequence of a 53010 polypeptide, e.g., alteringthe sequence, e.g., by substitution or deletion of one or more residuesof a non-conserved region, a domain or residue disclosed herein, andtesting the altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragmentor analog of a 53010 polypeptide a biological activity of a naturallyoccurring 53010 polypeptide. The method includes: altering the sequence,e.g., by substitution or deletion of one or more residues, of a 53010polypeptide, e.g., altering the sequence of a non-conserved region, or adomain or residue described herein, and testing the altered polypeptidefor the desired activity.

Anti-53010 Antibodies

In another aspect, the invention provides an anti-53010 antibody, or afragment thereof (e.g., an antigen-binding fragment thereof). The term“antibody” as used herein refers to an immunoglobulin molecule orimmunologically active portion thereof, i.e., an antigen-bindingportion. As used herein, the term “antibody” refers to a proteincomprising at least one, and preferably two, heavy (H) chain variableregions (abbreviated herein as VH), and at least one and preferably twolight (L) chain variable regions (abbreviated herein as VL). The VH andVL regions can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (“CDR”), interspersed withregions that are more conserved, termed “framework regions” (FR). Theextent of the framework region and CDR's has been precisely defined(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917, which are incorporated herein by reference). Each VH and VLis composed of three CDR's and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The anti-53010 antibody can further include a heavy and light chainconstant region, to thereby form a heavy and light immunoglobulin chain,respectively. In one embodiment, the antibody is a tetramer of two heavyimmunoglobulin chains and two light immunoglobulin chains, wherein theheavy and light immunoglobulin chains are inter-connected by, e.g.,disulfide bonds. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. The light chain constant region is comprisedof one domain, CL. The variable region of the heavy and light chainscontains a binding domain that interacts with an antigen. The constantregions of the antibodies typically mediate the binding of the antibodyto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (Clq) of the classicalcomplement system.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. The recognized human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 KDa or 214 amino acids) are encoded by a variableregion gene at the NH2-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH—terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

The term “antigen-binding fragment” of an antibody (or simply “antibodyportion,” or “fragment”), as used herein, refers to one or morefragments of a full-length antibody that retain the ability tospecifically bind to the antigen, e.g., 53010 polypeptide or fragmentthereof. Examples of antigen-binding fragments of the anti-53010antibody include, but are not limited to: (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also encompassed withinthe term “antigen-binding fragment” of an antibody. These antibodyfragments are obtained using conventional techniques known to those withskill in the art, and the fragments are screened for utility in the samemanner as are intact antibodies.

The anti-53010 antibody can be a polyclonal or a monoclonal antibody. Inother embodiments, the antibody can be recombinantly produced, e.g.,produced by phage display or by combinatorial methods.

Phage display and combinatorial methods for generating anti-53010antibodies are known in the art (as described in, e.g., Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO92/18619; Dower et al. International Publication No. WO 91/17271; Winteret al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Ladner et al. International Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contentsof all of which are incorporated by reference herein).

In one embodiment, the anti-53010 antibody is a fully human antibody(e.g., an antibody made in a mouse which has been genetically engineeredto produce an antibody from a human immunoglobulin sequence), or anon-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g.,monkey), camel antibody. Preferably, the non-human antibody is a rodent(mouse or rat antibody). Method of producing rodent antibodies are knownin the art.

Human monoclonal antibodies can be generated using transgenic micecarrying the human immunoglobulin genes rather than the mouse system.Splenocytes from these transgenic mice immunized with the antigen ofinterest are used to produce hybridomas that secrete human mAbs withspecific affinities for epitopes from a human protein (see, e.g., Woodet al. International Application WO 91/00906, Kucherlapati et al. PCTpublication WO 91/10741; Lonberg et al. International Application WO92/03918; Kay et al. International Application 92/03917; Lonberg, N. etal. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet.7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon etal. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol21:1323-1326).

An anti-53010 antibody can be one in which the variable region, or aportion thereof, e.g., the CDR's, are generated in a non-human organism,e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodiesare within the invention. Antibodies generated in a non-human organism,e.g., a rat or mouse, and then modified, e.g., in the variable frameworkor constant region, to decrease antigenicity in a human are within theinvention.

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art. For example, a gene encoding the Fc constant region of amurine (or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al. (1988 Science240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, JImmunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura etal., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two butgenerally all three recipient CDR's (of heavy and or light immuoglobulinchains) replaced with a donor CDR. The antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDR's may bereplaced with non-human CDR's. It is only necessary to replace thenumber of CDR's required for binding of the humanized antibody to a53010 or a fragment thereof. Preferably, the donor will be a rodentantibody, e.g., a rat or mouse antibody, and the recipient will be ahuman framework or a human consensus framework. Typically, theimmunoglobulin providing the CDR's is called the “donor” and theimmunoglobulin providing the framework is called the “acceptor.” In oneembodiment, the donor immunoglobulin is a non-human (e.g., rodent). Theacceptor framework is a naturally-occurring (e.g., a human) framework ora consensus framework, or a sequence about 85% or higher, preferably90%, 95%, 99% or higher identical thereto. As used herein, the term“consensus sequence” refers to the sequence formed from the mostfrequently occurring amino acids (or nucleotides) in a family of relatedsequences (See e.g., Winnaker, From Genes to Clones(Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins,each position in the consensus sequence is occupied by the amino acidoccurring most frequently at that position in the family. If two aminoacids occur equally frequently, either can be included in the consensussequence. A “consensus framework” refers to the framework region in theconsensus immunoglobulin sequence.

An antibody can be humanized by methods known in the art. Humanizedantibodies can be generated by replacing sequences of the Fv variableregion which are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison, S. L., 1985,Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and byQueen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S.Pat. No. 5,693,762, the contents of all of which are hereby incorporatedby reference. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from a hybridoma producing anantibody against a 53010 polypeptide or fragment thereof. Therecombinant DNA encoding the humanized antibody, or fragment thereof,can then be cloned into an appropriate expression vector.

Humanized or CDR-grafted antibodies can be produced by CDR-grafting orCDR substitution, wherein one, two, or all CDR's of an immunoglobulinchain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al.1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidleret al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539,the contents of all of which are hereby expressly incorporated byreference. Winter describes a CDR-grafting method which may be used toprepare the humanized antibodies of the present invention (UK PatentApplication GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No.5,225,539), the contents of which is expressly incorporated byreference.

Also within the scope of the invention are humanized antibodies in whichspecific amino acids have been substituted, deleted or added. Preferredhumanized antibodies have amino acid substitutions in the frameworkregion, such as to improve binding to the antigen. For example, ahumanized antibody will have framework residues identical to the donorframework residue or to another amino acid other than the recipientframework residue. To generate such antibodies, a selected, small numberof acceptor framework residues of the humanized immunoglobulin chain canbe replaced by the corresponding donor amino acids. Preferred locationsof the substitutions include amino acid residues adjacent to the CDR, orwhich are capable of interacting with a CDR (see e.g., U.S. Pat. No.5,585,089). Criteria for selecting amino acids from the donor aredescribed in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat.No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, thecontents of which are hereby incorporated by reference. Other techniquesfor humanizing antibodies are described in Padlan et al. EP 519596 A1,published on Dec. 23, 1992.

In preferred embodiments an antibody can be made by immunizing withpurified 53010 antigen, or a fragment thereof, e.g., a fragmentdescribed herein, tissue, e.g., crude tissue preparations, whole cells,preferably living cells, lysed cells, or cell fractions.

A full-length 53010 protein or, antigenic peptide fragment of 53010 canbe used as an immunogen or can be used to identify anti-53010 antibodiesmade with other immunogens, e.g., cells, membrane preparations, and thelike. The antigenic peptide of 53010 should include at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:2 andencompasses an epitope of 53010. Preferably, the antigenic peptideincludes at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues.

Fragments of 53010 which include residues from about amino acid 51 to80, from about 140 to 152, from about 232 to 248, and from about 410 to435 of SEQ ID NO:2 can be used to make, e.g., used as immunogens or usedto characterize the specificity of an antibody, antibodies againsthydrophilic regions of the 53010 protein. Similarly, fragments of 53010which include residues all or part of a hydrophilic sequence from aboutamino acid 190 to 205, from about 265 to 281, and from about 440 to 458of SEQ ID NO:2 can be used to make an antibody against a hydrophobicregion of the 53010 protein; a fragment of 53010 which include residuesabout 44 to 545, 219 to 234, or 125 to 135 can be used to make anantibody against the carboxylesterase region of the 53010 protein.

Antibodies reactive with, or specific for, any of these regions, orother regions or domains described herein are provided.

Antibodies which bind only native 53010 protein, only denatured orotherwise non-native 53010 protein, or which bind both, are with in theinvention. Antibodies with linear or conformational epitopes are withinthe invention. Conformational epitopes can sometimes be identified byidentifying antibodies which bind to native but not denatured 53010protein.

Preferred epitopes encompassed by the antigenic peptide are regions of53010 are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity. For example, anEmini surface probability analysis of the human 53010 protein sequencecan be used to indicate the regions that have a particularly highprobability of being localized to the surface of the 53010 protein andare thus likely to constitute surface residues useful for targetingantibody production.

In preferred embodiments antibodies can bind one or more of purifiedantigen, tissue, e.g., tissue sections, whole cells, preferably livingcells, lysed cells, or cell fractions.

The anti-53010 antibody can be a single chain antibody. A single-chainantibody (scFV) may be engineered (see, for example, Colcher, D. et al.(1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin CancerRes 2:245-52). The single chain antibody can be dimerized ormultimerized to generate multivalent antibodies having specificities fordifferent epitopes of the same target 53010 protein.

In a preferred embodiment the antibody has effector function and/or canfix complement. In other embodiments the antibody does not recruiteffector cells; or fix complement.

In a preferred embodiment, the antibody has reduced or no ability tobind an Fc receptor. For example, it is a isotype or subtype, fragmentor other mutant, which does not support binding to an Fc receptor, e.g.,it has a mutagenized or deleted Fc receptor binding region.

In a preferred embodiment, an anti-53010 antibody alters (e.g.,increases or decreases) the carboxylesterase activity of a 53010polypeptide. For example, the antibody can bind at or in proximity tothe active site, e.g., to an epitope that includes a residue locatedfrom about 125-135 of SEQ ID NO:2 or 219-234 of SEQ ID NO:2.

The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e.g.,ricin or diphtheria toxin or active fragment hereof, or a radioactivenucleus, or imaging agent, e.g. a radioactive, enzymatic, or other,e.g., imaging agent, e.g., a NMR contrast agent. Labels which producedetectable radioactive emissions or fluorescence are preferred.

An anti-53010 antibody (e.g., monoclonal antibody) can be used toisolate 53010 by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, an anti-53010 antibody can be used todetect 53010 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theprotein. Anti-53010 antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance (i.e., antibody labelling). Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

The invention also includes a nucleic acid which encodes an anti-53010antibody, e.g., an anti-53010 antibody described herein. Also includedare vectors which include the nucleic acid and cells transformed withthe nucleic acid, particularly cells which are useful for producing anantibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

The invention also includes cell lines, e.g., hybridomas, which make ananti-53010 antibody, e.g., an antibody described herein, and method ofusing said cells to make a 53010 antibody.

Recombinant Expression Vectors, Host Cells and Genetically EngineeredCells

In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a 53010 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Preferably therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or polypeptides, including fusion proteins or polypeptides,encoded by nucleic acids as described herein (e.g., 53010 proteins,mutant forms of 53010 proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of 53010 proteins in prokaryotic or eukaryotic cells. Forexample, polypeptides of the invention can be expressed in E. coli,insect cells (e.g., using baculovirus expression vectors), yeast cellsor mammalian cells. Suitable host cells are discussed further inGoeddel, (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

Purified fusion proteins can be used in 53010 activity assays, (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for 53010 proteins. In a preferredembodiment, a fusion protein expressed in a retroviral expression vectorof the present invention can be used to infect bone marrow cells whichare subsequently transplanted into irradiated recipients. The pathologyof the subject recipient is then examined after sufficient time haspassed (e.g., six weeks).

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman, S., (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.119-128). Another strategy is to alter the nucleic acid sequence of thenucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The 53010 expression vector can be a yeast expression vector, a vectorfor expression in insect cells, e.g., a baculovirus expression vector ora vector suitable for expression in mammalian cells.

When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40.

In another embodiment, the promoter is an inducible promoter, e.g., apromoter regulated by a steroid hormone, by a polypeptide hormone (e.g.,by means of a signal transduction pathway), or by a heterologouspolypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and“Tet-Off”; see, e.g., Clontech Inc., Calif., Gossen and Bujard (1992)Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human GeneTherapy 9:983).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example, the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a 53010 nucleic acidmolecule within a recombinant expression vector or a 53010 nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a53010 protein can be expressed in bacterial cells (such as E. coli),insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells (African green monkey kidney cells CV-1 originSV40 cells; Gluzman (1981) CellI23:175-182)). Other suitable host cellsare known to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell of the invention can be used to produce (i.e., express) a53010 protein. Accordingly, the invention further provides methods forproducing a 53010 protein using the host cells of the invention. In oneembodiment, the method includes culturing the host cell of the invention(into which a recombinant expression vector encoding a 53010 protein hasbeen introduced) in a suitable medium such that a 53010 protein isproduced. In another embodiment, the method further includes isolating a53010 protein from the medium or the host cell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a 53010 transgene, or which otherwisemisexpress 53010. The cell preparation can consist of human or non-humancells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, orpig cells. In preferred embodiments, the cell or cells include a 53010transgene, e.g., a heterologous form of a 53010, e.g., a gene derivedfrom humans (in the case of a non-human cell). The 53010 transgene canbe misexpressed, e.g., overexpressed or underexpressed. In otherpreferred embodiments, the cell or cells include a gene thatmis-expresses an endogenous 53010, e.g., a gene the expression of whichis disrupted, e.g., a knockout. Such cells can serve as a model forstudying disorders that are related to mutated or mis-expressed 53010alleles or for use in drug screening.

In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject 53010 polypeptide.

Also provided are cells, preferably human cells, e.g., humanhematopoietic or fibroblast cells, in which an endogenous 53010 is underthe control of a regulatory sequence that does not normally control theexpression of the endogenous 53010 gene. The expression characteristicsof an endogenous gene within a cell, e.g., a cell line or microorganism,can be modified by inserting a heterologous DNA regulatory element intothe genome of the cell such that the inserted regulatory element isoperably linked to the endogenous 53010 gene. For example, an endogenous53010 gene which is “transcriptionally silent,” e.g., not normallyexpressed, or expressed only at very low levels, may be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell. Techniquessuch as targeted homologous recombinations, can be used to insert theheterologous DNA as described in, e.g., Chappel, U.S. Pat. No.5,272,071; WO 91/06667, published in May 16, 1991.

In a preferred embodiment, recombinant cells described herein can beused for replacement therapy in a subject. For example, a nucleic acidencoding a 53010 polypeptide operably linked to an inducible promoter(e.g., a steroid hormone receptor-regulated promoter) is introduced intoa human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell.The cell is cultivated and encapsulated in a biocompatible material,such as poly-lysine alginate, and subsequently implanted into thesubject. See, e.g., Lanza (1996) Nat. Biotechnol 14:1107; Joki et al.(2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Productionof 53010 polypeptide can be regulated in the subject by administering anagent (e.g., a steroid hormone) to the subject. In another preferredembodiment, the implanted recombinant cells express and secrete anantibody specific for a 53010 polypeptide. The antibody can be anyantibody or any antibody derivative described herein.

Transgenic Animals

The invention provides non-human transgenic animals. Such animals areuseful for studying the function and/or activity of a 53010 protein andfor identifying and/or evaluating modulators of 53010 activity. As usedherein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA or arearrangement, e.g., a deletion of endogenous chromosomal DNA, whichpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A transgene can direct the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal, other transgenes, e.g., a knockout, reduce expression. Thus, atransgenic animal can be one in which an endogenous 53010 gene has beenaltered by, e.g., by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to atransgene of the invention to direct expression of a 53010 protein toparticular cells. A transgenic founder animal can be identified basedupon the presence of a 53010 transgene in its genome and/or expressionof 53010 mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding a53010 protein can further be bred to other transgenic animals carryingother transgenes.

53010 proteins or polypeptides can be expressed in transgenic animals orplants, e.g., a nucleic acid encoding the protein or polypeptide can beintroduced into the genome of an animal. In preferred embodiments thenucleic acid is placed under the control of a tissue specific promoter,e.g., a milk or egg specific promoter, and recovered from the milk oreggs produced by the animal. Suitable animals are mice, pigs, cows,goats, and sheep.

The invention also includes a population of cells from a transgenicanimal, as discussed, e.g., below.

Uses

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Inaddition, the 53010 polypeptides of the invention are useful in vitrofor the chemical synthesis and/or modification of chemical compounds(e.g., for the stereospecific synthesis and hydrolysis of esters).

The isolated nucleic acid molecules of the invention can be used, forexample, to express a 53010 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect a 53010mRNA (e.g., in a biological sample) or a genetic alteration in a 53010gene, and to modulate 53010 activity, as described further below. The53010 proteins can be used to treat disorders characterized byinsufficient or excessive production of a 53010 substrate or productionof 53010 inhibitors. In addition, the 53010 proteins can be used toscreen for naturally occurring 53010 substrates, to screen for drugs orcompounds which modulate 53010 activity, as well as to treat disorderscharacterized by insufficient or excessive production of 53010 proteinor production of 53010 protein forms which have decreased, aberrant orunwanted activity compared to 53010 wild type protein (e.g., pain orpain-related disorders). Moreover, the anti-53010 antibodies of theinvention can be used to detect and isolate 53010 proteins, regulate thebioavailability of 53010 proteins, and modulate 53010 activity.

A method of evaluating a compound for the ability to interact with,e.g., bind, a subject 53010 polypeptide is provided. The methodincludes: contacting the compound with the subject 53010 polypeptide;and evaluating ability of the compound to interact with, e.g., to bindor form a complex with the subject 53010 polypeptide. This method can beperformed in vitro, e.g., in a cell free system, or in vivo, e.g., in atwo-hybrid interaction trap assay. This method can be used to identifynaturally occurring molecules that interact with subject 53010polypeptide. It can also be used to find natural or synthetic inhibitorsof subject 53010 polypeptide. Screening methods are discussed in moredetail below.

Screening Assays

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to 53010 proteins, have astimulatory or inhibitory effect on, for example, 53010 expression or53010 activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a 53010 substrate. Compounds thusidentified can be used to modulate the activity of target gene products(e.g., 53010 genes) in a therapeutic protocol, to elaborate thebiological function of the target gene product, or to identify compoundsthat disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a 53010 protein or polypeptideor a biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compounds thatbind to or modulate an activity of a 53010 protein or polypeptide or abiologically active portion thereof.

A carboxylesterase activity of a 53010 polypeptide can be detected by anin vitro hydrolase activity assay well known in the art and can befound, for example, in Newcomb et al. (1997) Proc. Natl Acad. Sci. USA94:7464-7468, Newcomb et al. (1996) Insect Biochem. Mol. Biol. 27:15-25,and Campbell et al. (1997) Biochem. Genet. 53:17-40. These assaysinclude, but are not limited to, the disappearance of a substrate, orappearance of a product, e.g., by spectrophotometric measurement ofartificial ester substrates, such as naphthyl acetate (NA),p-nitrophenyl acetate (p-NPA), and methylthiobutyrate (MtB), or by aradiometric measurement of labelled substrates, such as ¹⁴C-labelledesters.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al.(1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994)Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991)J. Mol. Biol. 222:301-310;Ladner supra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a 53010 protein or biologically active portion thereof iscontacted with a test compound, and the ability of the test compound tomodulate 53010 activity is determined. Determining the ability of thetest compound to modulate 53010 activity can be accomplished bymonitoring, for example, hydrolysis of carboxyl group. The cell, forexample, can be of mammalian origin, e.g., human.

The ability of the test compound to modulate 53010 binding to acompound, e.g., a 53010 substrate, or to bind to 53010 can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to 53010 can bedetermined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, 53010 could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulate53010 binding to a 53010 substrate in a complex. For example, compounds(e.g., 53010 substrates) can be labeled with 125I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

The ability of a compound (e.g., a 53010 substrate) to interact with53010 with or without the labeling of any of the interactants can beevaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with 53010 without the labeling of either thecompound or the 53010. McConnell, H. M. et al. (1992) Science257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a compound and 53010.

In yet another embodiment, a cell-free assay is provided in which a53010 protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to bind to the 53010protein or biologically active portion thereof is evaluated. Preferredbiologically active portions of the 53010 proteins to be used in assaysof the present invention include fragments which participate ininteractions with non-53010 molecules, e.g., fragments with high surfaceprobability scores. Soluble and/or membrane-bound forms of isolatedproteins (e.g., 53010 proteins or biologically active portions thereof)can be used in the cell-free assays of the invention. Whenmembrane-bound forms of the protein are used, it may be desirable toutilize a solubilizing agent. Examples of such solubilizing agentsinclude non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl═N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the 53010 protein tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either 53010, an anti-53010 antibodyor its target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a53010 protein, or interaction of a 53010 protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/53010 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or 53010 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of 53010binding or activity determined using standard techniques.

Other techniques for immobilizing either a 53010 protein or a targetmolecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated 53010 protein or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith 53010 protein or target molecules but which do not interfere withbinding of the 53010 protein to its target molecule. Such antibodies canbe derivatized to the wells of the plate, and unbound target or 53010protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the 53010 protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the 53010 protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas, G.,and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography(gel filtration chromatography, ion-exchange chromatography);electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocolsin Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation(see, for example, Ausubel, F. et al., eds. (1999) Current Protocols inMolecular Biology, J. Wiley: New York). Such resins and chromatographictechniques are known to one skilled in the art (see, e.g., Heegaard, N.H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997)J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescenceenergy transfer may also be conveniently utilized, as described herein,to detect binding without further purification of the complex fromsolution.

In a preferred embodiment, the assay includes contacting the 53010protein or biologically active portion thereof with a known compoundwhich binds 53010 to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a 53010 protein, wherein determining the ability of thetest compound to interact with a 53010 protein includes determining theability of the test compound to preferentially bind to 53010 orbiologically active portion thereof, or to modulate the activity of atarget molecule, as compared to the known compound.

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.For the purposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target gene product. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and small molecules.The preferred target genes/products for use in this embodiment are the53010 genes herein identified. In an alternative embodiment, theinvention provides methods for determining the ability of the testcompound to modulate the activity of a 53010 protein through modulationof the activity of a downstream effector of a 53010 target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined, or the binding of the effector to an appropriatetarget can be determined, as previously described.

To identify compounds that interfere with the interaction between thetarget gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form complex. In order to testan inhibitory agent, the reaction mixture is provided in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene product and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene product and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target gene product can also be compared to complex formationwithin reaction mixtures containing the test compound and mutant targetgene product. This comparison can be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target gene product orthe binding partner onto a solid phase, and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the targetgene products and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface (e.g., a microtiter plate), while the non-anchoredspecies is labeled, either directly or indirectly. The anchored speciescan be immobilized by non-covalent or covalent attachments.Alternatively, an immobilized antibody specific for the species to beanchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface,e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target gene product andthe interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

In yet another aspect, the 53010 proteins can be used as “bait proteins”in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), to identify other proteins, which bind to or interact with53010 (“53010-binding proteins” or “53010-bp”) and are involved in 53010activity. Such 53010-bps can be activators or inhibitors of signals bythe 53010 proteins or 53010 targets as, for example, downstream elementsof a 53010-mediated signaling pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 53010 protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 53010 protein can bethe fused to the activator domain.) If the “bait” and the “prey”proteins are able to interact, in vivo, forming a 53010-dependentcomplex, the DNA-binding and activation domains of the transcriptionfactor are brought into close proximity. This proximity allowstranscription of a reporter gene (e.g., lacZ) which is operably linkedto a transcriptional regulatory site responsive to the transcriptionfactor. Expression of the reporter gene can be detected and cellcolonies containing the functional transcription factor can be isolatedand used to obtain the cloned gene which encodes the protein whichinteracts with the 53010 protein.

In another embodiment, modulators of 53010 expression are identified.For example, a cell or cell free mixture is contacted with a candidatecompound and the expression of 53010 mRNA or protein evaluated relativeto the level of expression of 53010 mRNA or protein in the absence ofthe candidate compound. When expression of 53010 mRNA or protein isgreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of 53010 mRNA orprotein expression. Alternatively, when expression of 53010 mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of 53010 mRNA or protein expression. Thelevel of 53010 mRNA or protein expression can be determined by methodsdescribed herein for detecting 53010 mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a 53010 protein can beconfirmed in vivo, e.g., in an animal such as an animal model forlacking ability of hydrolysis of carboxylic esters.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 53010 modulating agent, an antisense 53010 nucleic acidmolecule, a 53010-specific antibody, or a 53010-binding partner) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

Detection Assays

Portions or fragments of the nucleic acid sequences identified hereincan be used as polynucleotide reagents. For example, these sequences canbe used to: (i) map their respective genes on a chromosome e.g., tolocate gene regions associated with genetic disease or to associate53010 with a disease; (ii) identify an individual from a minutebiological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

Chromosome Mapping

The 53010 nucleotide sequences or portions thereof can be used to mapthe location of the 53010 genes on a chromosome. This process is calledchromosome mapping. Chromosome mapping is useful in correlating the53010 sequences with genes associated with disease.

Briefly, 53010 genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the 53010 nucleotidesequences. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the 53010 sequences willyield an amplified fragment.

A panel of somatic cell hybrids in which each cell line contains eithera single human chromosome or a small number of human chromosomes, and afull set of mouse chromosomes, can allow easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924).

Other mapping strategies e.g., in situ hybridization (described in Fan,Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries can be used to map 53010 to achromosomal location.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al., Human Chromosomes: A Manual of BasicTechniques ((1988) Pergamon Press, New York).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the 53010 gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

Tissue Typing

53010 sequences can be used to identify individuals from biologicalsamples using, e.g., restriction fragment length polymorphism (RFLP). Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, the fragments separated, e.g., in a Southern blot,and probed to yield bands for identification. The sequences of thepresent invention are useful as additional DNA markers for RFLP(described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can also be used todetermine the actual base-by-base DNA sequence of selected portions ofan individual's genome. Thus, the 53010 nucleotide sequences describedherein can be used to prepare two PCR primers from the 5′ and 3′ ends ofthe sequences. These primers can then be used to amplify an individual'sDNA and subsequently sequence it. Panels of corresponding DNA sequencesfrom individuals, prepared in this manner, can provide unique individualidentifications, as each individual will have a unique set of such DNAsequences due to allelic differences.

Allelic variation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. Each of thesequences described herein can, to some degree, be used as a standardagainst which DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can provide positiveindividual identification with a panel of perhaps 10 to 1,000 primerswhich each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:3 are used, amore appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from 53010 nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

Use of Partial 53010 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 (e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 having a length of at least 20bases, preferably at least 30 bases) are particularly appropriate forthis use.

The 53010 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such 53010 probes can be used to identify tissue by speciesand/or by organ type.

In a similar fashion, these reagents, e.g., 53010 primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual.

Generally, the invention provides, a method of determining if a subjectis at risk for a disorder related to a lesion in or the misexpression ofa gene which encodes 53010.

Such disorders include, e.g., a disorder associated with themisexpression of 53010 gene; a disorder a disorder involving aberrant ordeficient hydrolysis of carboxylic esters; pain or pain-relateddisorders; and a disorder of the blood coagulation system.

The method includes one or more of the following:

detecting, in a tissue of the subject, the presence or absence of amutation which affects the expression of the 53010 gene, or detectingthe presence or absence of a mutation in a region which controls theexpression of the gene, e.g., a mutation in the 5′ control region;

detecting, in a tissue of the subject, the presence or absence of amutation which alters the structure of the 53010 gene;

detecting, in a tissue of the subject, the misexpression of the 53010gene, at the mRNA level, e.g., detecting a non-wild type level of amRNA;

detecting, in a tissue of the subject, the misexpression of the gene, atthe protein level, e.g., detecting a non-wild type level of a 53010polypeptide.

In preferred embodiments the method includes: ascertaining the existenceof at least one of: a deletion of one or more nucleotides from the 53010gene; an insertion of one or more nucleotides into the gene, a pointmutation, e.g., a substitution of one or more nucleotides of the gene, agross chromosomal rearrangement of the gene, e.g., a translocation,inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequencefrom SEQ ID NO:1, or naturally occurring mutants thereof or 5′ or 3′flanking sequences naturally associated with the 53010 gene; (ii)exposing the probe/primer to nucleic acid of the tissue; and detecting,by hybridization, e.g., in situ hybridization, of the probe/primer tothe nucleic acid, the presence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 53010 gene; the presence of anon-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of 53010.

Methods of the invention can be used prenatally or to determine if asubject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structureof a 53010 gene, an abnormal structure being indicative of risk for thedisorder.

In preferred embodiments the method includes contacting a sample fromthe subject with an antibody to the 53010 protein or a nucleic acid,which hybridizes specifically with the gene. These and other embodimentsare discussed below.

Diagnostic and Prognostic Assays

Diagnostic and prognostic assays of the invention include method forassessing the expression level of 53010 molecules and for identifyingvariations and mutations in the sequence of 53010 molecules.

Expression Monitoring and Profiling

The presence, level, or absence of 53010 protein or nucleic acid in abiological sample can be evaluated by obtaining a biological sample froma test subject and contacting the biological sample with a compound oran agent capable of detecting 53010 protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes 53010 protein such that the presence of 53010protein or nucleic acid is detected in the biological sample. The term“biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum. The level ofexpression of the 53010 gene can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the 53010genes; measuring the amount of protein encoded by the 53010 genes; ormeasuring the activity of the protein encoded by the 53010 genes.

The level of mRNA corresponding to the 53010 gene in a cell can bedetermined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length 53010 nucleic acid, suchas the nucleic acid of SEQ ID NO: 1, or a portion thereof, such as anoligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to 53010 mRNA or genomic DNA. The probe can be disposed on anaddress of an array, e.g., an array described below. Other suitableprobes for use in the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array described below. A skilledartisan can adapt known mRNA detection methods for use in detecting thelevel of mRNA encoded by the 53010 genes.

The level of mRNA in a sample that is encoded by one of 53010 can beevaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987)U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh et al., (1989), Proc. Natl.Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988)Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S.Pat. No. 5,854,033) or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniquesknown in the art. As used herein, amplification primers are defined asbeing a pair of nucleic acid molecules that can anneal to 5′ or 3′regions of a gene (plus and minus strands, respectively, or vice-versa)and contain a short region in between. In general, amplification primersare from about 10 to 30 nucleotides in length and flank a region fromabout 50 to 200 nucleotides in length. Under appropriate conditions andwith appropriate reagents, such primers permit the amplification of anucleic acid molecule comprising the nucleotide sequence flanked by theprimers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes the 53010gene being analyzed.

In another embodiment, the methods further contacting a control samplewith a compound or agent capable of detecting 53010 mRNA, or genomicDNA, and comparing the presence of 53010 mRNA or genomic DNA in thecontrol sample with the presence of 53010 mRNA or genomic DNA in thetest sample. In still another embodiment, serial analysis of geneexpression, as described in U.S. Pat. No. 5,695,937, is used to detect53010 transcript levels.

A variety of methods can be used to determine the level of proteinencoded by 53010. In general, these methods include contacting an agentthat selectively binds to the protein, such as an antibody with asample, to evaluate the level of protein in the sample. In a preferredembodiment, the antibody bears a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with a detectablesubstance. Examples of detectable substances are provided herein.

The detection methods can be used to detect 53010 protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of 53010 protein include enzyme linked immunosorbent assays(ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay(EIA), radioimmunoassay (RIA), and Western blot analysis. In vivotechniques for detection of 53010 protein include introducing into asubject a labeled anti-53010 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. In anotherembodiment, the sample is labeled, e.g., biotinylated and then contactedto the antibody, e.g., an anti-53010 antibody positioned on an antibodyarray (as described below). The sample can be detected, e.g., withavidin coupled to a fluorescent label.

In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 53010protein, and comparing the presence of 53010 protein in the controlsample with the presence of 53010 protein in the test sample.

The invention also includes kits for detecting the presence of 53010 ina biological sample. For example, the kit can include a compound oragent capable of detecting 53010 protein or mRNA in a biological sample;and a standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect 53010 protein or nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a polypeptidecorresponding to a marker of the invention; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 53010 expression or activity. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as pain or deregulated cellproliferation.

In one embodiment, a disease or disorder associated with aberrant orunwanted 53010 expression or activity is identified. A test sample isobtained from a subject and 53010 protein or nucleic acid (e.g., mRNA orgenomic DNA) is evaluated, wherein the level, e.g., the presence orabsence, of 53010 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted 53010 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest, including a biological fluid (e.g., serum), cell sample, ortissue.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with aberrantor unwanted 53010 expression or activity. For example, such methods canbe used to determine whether a subject can be effectively treated withan agent for pain or a pain-related disorder.

In another aspect, the invention features a computer medium having aplurality of digitally encoded data records. Each data record includes avalue representing the level of expression of 53010 in a sample, and adescriptor of the sample. The descriptor of the sample can be anidentifier of the sample, a subject from which the sample was derived(e.g., a patient), a diagnosis, or a treatment (e.g., a preferredtreatment). In a preferred embodiment, the data record further includesvalues representing the level of expression of genes other than 53010(e.g., other genes associated with a 53010-disorder, or other genes onan array). The data record can be structured as a table, e.g., a tablethat is part of a database such as a relational database (e.g., a SQLdatabase of the Oracle or Sybase database environments).

Also featured is a method of evaluating a sample. The method includesproviding a sample, e.g., from the subject, and determining a geneexpression profile of the sample, wherein the profile includes a valuerepresenting the level of 53010 expression. The method can furtherinclude comparing the value or the profile (i.e., multiple values) to areference value or reference profile. The gene expression profile of thesample can be obtained by any of the methods described herein (e.g., byproviding a nucleic acid from the sample and contacting the nucleic acidto an array). The method can be used to diagnose a pain-related disorderin a subject wherein an unwanted 53010 expression is an indication thatthe subject has or is disposed to having a pain-related disorder. Themethod can be used to monitor a treatment for a pain-related disorder ina subject. For example, the gene expression profile can be determinedfor a sample from a subject undergoing treatment. The profile can becompared to a reference profile or to a profile obtained from thesubject prior to treatment or prior to onset of the disorder (see, e.g.,Golub et al. (1999) Science 286:531).

In yet another aspect, the invention features a method of evaluating atest compound (see also, “Screening Assays”, above). The method includesproviding a cell and a test compound; contacting the test compound tothe cell; obtaining a subject expression profile for the contacted cell;and comparing the subject expression profile to one or more referenceprofiles. The profiles include a value representing the level of 53010expression. In a preferred embodiment, the subject expression profile iscompared to a target profile, e.g., a profile for a normal cell or fordesired condition of a cell. The test compound is evaluated favorably ifthe subject expression profile is more similar to the target profilethan an expression profile obtained from an uncontacted cell.

In another aspect, the invention features, a method of evaluating asubject. The method includes: a) obtaining a sample from a subject,e.g., from a caregiver, e.g., a caregiver who obtains the sample fromthe subject; b) determining a subject expression profile for the sample.Optionally, the method further includes either or both of steps: c)comparing the subject expression profile to one or more referenceexpression profiles; and d) selecting the reference profile most similarto the subject reference profile. The subject expression profile and thereference profiles include a value representing the level of 53010expression. A variety of routine statistical measures can be used tocompare two reference profiles. One possible metric is the length of thedistance vector that is the difference between the two profiles. Each ofthe subject and reference profile is represented as a multi-dimensionalvector, wherein each dimension is a value in the profile.

The method can further include transmitting a result to a caregiver. Theresult can be the subject expression profile, a result of a comparisonof the subject expression profile with another profile, a most similarreference profile, or a descriptor of any of the aforementioned. Theresult can be transmitted across a computer network, e.g., the resultcan be in the form of a computer transmission, e.g., a computer datasignal embedded in a carrier wave.

Also featured is a computer medium having executable code for effectingthe following steps: receive a subject expression profile; access adatabase of reference expression profiles; and either i) select amatching reference profile most similar to the subject expressionprofile or ii) determine at least one comparison score for thesimilarity of the subject expression profile to at least one referenceprofile. The subject expression profile, and the reference expressionprofiles each include a value representing the level of 53010expression.

Arrays and Uses Thereof

In another aspect, the invention features an array that includes asubstrate having a plurality of addresses. At least one address of theplurality includes a capture probe that binds specifically to a 53010molecule (e.g., a 53010 nucleic acid or a 53010 polypeptide). The arraycan have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000,or 10,000 or more addresses/cm², and ranges between. In a preferredembodiment, the plurality of addresses includes at least 10, 100, 500,1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, theplurality of addresses includes equal to or less than 10, 100, 500,1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be atwo-dimensional substrate such as a glass slide, a wafer (e.g., silicaor plastic), a mass spectroscopy plate, or a three-dimensional substratesuch as a gel pad. Addresses in addition to address of the plurality canbe disposed on the array.

In a preferred embodiment, at least one address of the pluralityincludes a nucleic acid capture probe that hybridizes specifically to a53010 nucleic acid, e.g., the sense or anti-sense strand. In onepreferred embodiment, a subset of addresses of the plurality ofaddresses has a nucleic acid capture probe for 53010. Each address ofthe subset can include a capture probe that hybridizes to a differentregion of a 53010 nucleic acid. In another preferred embodiment,addresses of the subset include a capture probe for a 53010 nucleicacid. Each address of the subset is unique, overlapping, andcomplementary to a different variant of 53010 (e.g., an allelic variant,or all possible hypothetical variants). The array can be used tosequence 53010 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

An array can be generated by various methods, e.g., by photolithographicmethods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681),mechanical methods (e.g., directed-flow methods as described in U.S.Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat.No. 5,288,514), and bead-based techniques (e.g., as described in PCTUS/93/04145).

In another preferred embodiment, at least one address of the pluralityincludes a polypeptide capture probe that binds specifically to a 53010polypeptide or fragment thereof. The polypeptide can be anaturally-occurring interaction partner of 53010 polypeptide.Preferably, the polypeptide is an antibody, e.g., an antibody describedherein (see “Anti-53010 Antibodies,” above), such as a monoclonalantibody or a single-chain antibody.

In another aspect, the invention features a method of analyzing theexpression of 53010. The method includes providing an array as describedabove; contacting the array with a sample and detecting binding of a53010-molecule (e.g., nucleic acid or polypeptide) to the array. In apreferred embodiment, the array is a nucleic acid array. Optionally themethod further includes amplifying nucleic acid from the sample prior orduring contact with the array.

In another embodiment, the array can be used to assay gene expression ina tissue to ascertain tissue specificity of genes in the array,particularly the expression of 53010. If a sufficient number of diversesamples is analyzed, clustering (e.g., hierarchical clustering, k-meansclustering, Bayesian clustering and the like) can be used to identifyother genes which are co-regulated with 53010. For example, the arraycan be used for the quantitation of the expression of multiple genes.Thus, not only tissue specificity, but also the level of expression of abattery of genes in the tissue is ascertained. Quantitative data can beused to group (e.g., cluster) genes on the basis of their tissueexpression per se and level of expression in that tissue.

For example, array analysis of gene expression can be used to assess theeffect of cell—cell interactions on 53010 expression. A first tissue canbe perturbed and nucleic acid from a second tissue that interacts withthe first tissue can be analyzed. In this context, the effect of onecell type on another cell type in response to a biological stimulus canbe determined, e.g., to monitor the effect of cell—cell interaction atthe level of gene expression.

In another embodiment, cells are contacted with a therapeutic agent. Theexpression profile of the cells is determined using the array, and theexpression profile is compared to the profile of like cells notcontacted with the agent. For example, the assay can be used todetermine or analyze the molecular basis of an undesirable effect of thetherapeutic agent. If an agent is administered therapeutically to treatone cell type but has an undesirable effect on another cell type, theinvention provides an assay to determine the molecular basis of theundesirable effect and thus provides the opportunity to co-administer acounteracting agent or otherwise treat the undesired effect. Similarly,even within a single cell type, undesirable biological effects can bedetermined at the molecular level. Thus, the effects of an agent onexpression of other than the target gene can be ascertained andcounteracted.

In another embodiment, the array can be used to monitor expression ofone or more genes in the array with respect to time. For example,samples obtained from different time points can be probed with thearray. Such analysis can identify and/or characterize the development ofa 53010-associated disease or disorder; and processes, such as acellular transformation associated with a 53010-associated disease ordisorder. The method can also evaluate the treatment and/or progressionof a 53010-associated disease or disorder

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including 53010) that could serve asa molecular target for diagnosis or therapeutic intervention.

In another aspect, the invention features an array having a plurality ofaddresses. Each address of the plurality includes a unique polypeptide.At least one address of the plurality has disposed thereon a 53010polypeptide or fragment thereof. Methods of producing polypeptide arraysare described in the art, e.g., in De Wildt et al. (2000). NatureBiotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270,103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G.,and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1.In a preferred embodiment, each addresses of the plurality has disposedthereon a polypeptide at least 60, 70, 80, 85, 90, 95 or 99% identicalto a 53010 polypeptide or fragment thereof. For example, multiplevariants of a 53010 polypeptide (e.g., encoded by allelic variants,site-directed mutants, random mutants, or combinatorial mutants) can bedisposed at individual addresses of the plurality. Addresses in additionto the address of the plurality can be disposed on the array.

The polypeptide array can be used to detect a 53010 binding compound,e.g., an antibody in a sample from a subject with specificity for a53010 polypeptide or the presence of a 53010-binding protein or ligand.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells (e.g., ascertaining the effect of 53010 expression onthe expression of other genes). This provides, for example, for aselection of alternate molecular targets for therapeutic intervention ifthe ultimate or downstream target cannot be regulated.

In another aspect, the invention features a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express 53010 or from a cell or subject in whicha 53010 mediated response has been elicited, e.g., by contact of thecell with 53010 nucleic acid or protein, or administration to the cellor subject 53010 nucleic acid or protein; providing a two dimensionalarray having a plurality of addresses, each address of the pluralitybeing positionally distinguishable from each other address of theplurality, and each address of the plurality having a unique captureprobe, e.g., wherein the capture probes are from a cell or subject whichdoes not express 53010 (or does not express as highly as in the case ofthe 53010 positive plurality of capture probes) or from a cell orsubject which in which a 53010 mediated response has not been elicited(or has been elicited to a lesser extent than in the first sample);contacting the array with one or more inquiry probes (which ispreferably other than a 53010 nucleic acid, polypeptide, or antibody),and thereby evaluating the plurality of capture probes. Binding, e.g.,in the case of a nucleic acid, hybridization with a capture probe at anaddress of the plurality, is detected, e.g., by signal generated from alabel attached to the nucleic acid, polypeptide, or antibody.

In another aspect, the invention features a method of analyzing aplurality of probes or a sample. The method is useful, e.g., foranalyzing gene expression. The method includes: providing a twodimensional array having a plurality of addresses, each address of theplurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, contacting the array with afirst sample from a cell or subject which express or mis-express 53010or from a cell or subject in which a 53010-mediated response has beenelicited, e.g., by contact of the cell with 53010 nucleic acid orprotein, or administration to the cell or subject 53010 nucleic acid orprotein; providing a two dimensional array having a plurality ofaddresses, each address of the plurality being positionallydistinguishable from each other address of the plurality, and eachaddress of the plurality having a unique capture probe, and contactingthe array with a second sample from a cell or subject which does notexpress 53010 (or does not express as highly as in the case of the 53010positive plurality of capture probes) or from a cell or subject which inwhich a 53010 mediated response has not been elicited (or has beenelicited to a lesser extent than in the first sample); and comparing thebinding of the first sample with the binding of the second sample.Binding, e.g., in the case of a nucleic acid, hybridization with acapture probe at an address of the plurality, is detected, e.g., bysignal generated from a label attached to the nucleic acid, polypeptide,or antibody. The same array can be used for both samples or differentarrays can be used. If different arrays are used the plurality ofaddresses with capture probes should be present on both arrays.

In another aspect, the invention features a method of analyzing 53010,e.g., analyzing structure, function, or relatedness to other nucleicacid or amino acid sequences. The method includes: providing a 53010nucleic acid or amino acid sequence; comparing the 53010 sequence withone or more preferably a plurality of sequences from a collection ofsequences, e.g., a nucleic acid or protein sequence database; to therebyanalyze 53010.

Detection of Variations or Mutations

The methods of the invention can also be used to detect geneticalterations in a 53010 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation in53010 protein activity or nucleic acid expression, such as pain or apain-related disorder. In preferred embodiments, the methods includedetecting, in a sample from the subject, the presence or absence of agenetic alteration characterized by at least one of an alterationaffecting the integrity of a gene encoding a 53010-protein, or themis-expression of the 53010 gene. For example, such genetic alterationscan be detected by ascertaining the existence of at least one of 1) adeletion of one or more nucleotides from a 53010 gene; 2) an addition ofone or more nucleotides to a 53010 gene; 3) a substitution of one ormore nucleotides of a 53010 gene, 4) a chromosomal rearrangement of a53010 gene; 5) an alteration in the level of a messenger RNA transcriptof a 53010 gene, 6) aberrant modification of a 53010 gene, such as ofthe methylation pattern of the genomic DNA, 7) the presence of anon-wild type splicing pattern of a messenger RNA transcript of a 53010gene, 8) a non-wild type level of a 53010-protein, 9) allelic loss of a53010 gene, and 10) inappropriate post-translational modification of a53010-protein.

An alteration can be detected without a probe/primer in a polymerasechain reaction, such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR), the latter of which can be particularlyuseful for detecting point mutations in the 53010-gene. This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a 53010 gene under conditions such thathybridization and amplification of the 53010-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.Alternatively, other amplification methods described herein or known inthe art can be used.

In another embodiment, mutations in a 53010 gene from a sample cell canbe identified by detecting alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined, e.g., by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in 53010 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA,two-dimensional arrays, e.g., chip based arrays. Such arrays include aplurality of addresses, each of which is positionally distinguishablefrom the other. A different probe is located at each address of theplurality. A probe can be complementary to a region of a 53010 nucleicacid or a putative variant (e.g., allelic variant) thereof. A probe canhave one or more mismatches to a region of a 53010 nucleic acid (e.g., adestabilizing mismatch). The arrays can have a high density ofaddresses, e.g., can contain hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in 53010 can be identified in two-dimensional arrayscontaining light-generated DNA probes as described in Cronin, M. T. etal. supra. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the 53010 gene anddetect mutations by comparing the sequence of the sample 53010 with thecorresponding wild-type (control) sequence. Automated sequencingprocedures can be utilized when performing the diagnostic assays ((1995)Biotechniques 19:448), including sequencing by mass spectrometry.

Other methods for detecting mutations in the 53010 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleebaet al. (1992) Methods Enzymol. 217:286-295).

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in 53010 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S.Pat. No. 5,459,039).

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in 53010 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control53010 nucleic acids will be denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence, the resulting alteration in electrophoretic mobility enablesthe detection of even a single base change. The DNA fragments may belabeled or detected with labeled probes. The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension (Saiki et al. (1986) Nature324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Afurther method of detecting point mutations is the chemical ligation ofoligonucleotides as described in Xu et al. ((2001) Nature Biotechnol.19:148). Adjacent oligonucleotides, one of which selectively anneals tothe query site, are ligated together if the nucleotide at the query siteof the sample nucleic acid is complementary to the queryoligonucleotide; ligation can be monitored, e.g., by fluorescent dyescoupled to the oligonucleotides.

Alternatively, allele specific amplification technology that depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

In another aspect, the invention features a set of oligonucleotides. Theset includes a plurality of oligonucleotides, each of which is at leastpartially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%,95%, 97%, 98%, or 99% complementary) to a 53010 nucleic acid.

In a preferred embodiment the set includes a first and a secondoligonucleotide. The first and second oligonucleotide can hybridize tothe same or to different locations of SEQ ID NO:1 or the complement ofSEQ ID NO: 1. Different locations can be different but overlapping, ornon-overlapping on the same strand. The first and second oligonucleotidecan hybridize to sites on the same or on different strands.

The set can be useful, e.g., for identifying SNP's, or identifyingspecific alleles of 53010. In a preferred embodiment, eacholigonucleotide of the set has a different nucleotide at aninterrogation position. In one embodiment, the set includes twooligonucleotides, each complementary to a different allele at a locus,e.g., a biallelic or polymorphic locus.

In another embodiment, the set includes four oligonucleotides, eachhaving a different nucleotide (e.g., adenine, guanine, cytosine, orthymidine) at the interrogation position. The interrogation position canbe a SNP or the site of a mutation. In another preferred embodiment, theoligonucleotides of the plurality are identical in sequence to oneanother (except for differences in length). The oligonucleotides can beprovided with differential labels, such that an oligonucleotide thathybridizes to one allele provides a signal that is distinguishable froman oligonucleotide that hybridizes to a second allele. In still anotherembodiment, at least one of the oligonucleotides of the set has anucleotide change at a position in addition to a query position, e.g., adestabilizing mutation to decrease the Tm of the oligonucleotide. Inanother embodiment, at least one oligonucleotide of the set has anon-natural nucleotide, e.g., inosine. In a preferred embodiment, theoligonucleotides are attached to a solid support, e.g., to differentaddresses of an array or to different beads or nanoparticles.

In a preferred embodiment the set of oligo nucleotides can be used tospecifically amplify, e.g., by PCR, or detect, a 53010 nucleic acid.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a 53010 gene.

Use of 53010 Molecules as Surrogate Markers

The 53010 molecules of the invention are also useful as markers ofdisorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the 53010 molecules of the invention may be detected,and may be correlated with one or more biological states in vivo. Forexample, the 53010 molecules of the invention may serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease may be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection may be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

The 53010 molecules of the invention are also useful as pharmacodynamicmarkers. As used herein, a “pharmacodynamic marker” is an objectivebiochemical marker which correlates specifically with drug effects. Thepresence or quantity of a pharmacodynamic marker is not related to thedisease state or disorder for which the drug is being administered;therefore, the presence or quantity of the marker is indicative of thepresence or activity of the drug in a subject. For example, apharmacodynamic marker may be indicative of the concentration of thedrug in a biological tissue, in that the marker is either expressed ortranscribed or not expressed or transcribed in that tissue inrelationship to the level of the drug. In this fashion, the distributionor uptake of the drug may be monitored by the pharmacodynamic marker.Similarly, the presence or quantity of the pharmacodynamic marker may berelated to the presence or quantity of the metabolic product of a drug,such that the presence or quantity of the marker is indicative of therelative breakdown rate of the drug in vivo. Pharmacodynamic markers areof particular use in increasing the sensitivity of detection of drugeffects, particularly when the drug is administered in low doses. Sinceeven a small amount of a drug may be sufficient to activate multiplerounds of marker (e.g., a 53010 marker) transcription or expression, theamplified marker may be in a quantity which is more readily detectablethan the drug itself. Also, the marker may be more easily detected dueto the nature of the marker itself; for example, using the methodsdescribed herein, anti-53010 antibodies may be employed in animmune-based detection system for a 53010 protein marker, or53010-specific radiolabeled probes may be used to detect a 53010 mRNAmarker. Furthermore, the use of a pharmacodynamic marker may offermechanism-based prediction of risk due to drug treatment beyond therange of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

The 53010 molecules of the invention are also useful as pharmacogenomicmarkers. As used herein, a “pharmacogenomic marker” is an objectivebiochemical marker which correlates with a specific clinical drugresponse or susceptibility in a subject (see, e.g., McLeod et al. (1999)Eur. J. Cancer 35:1650-1652). The presence or quantity of thepharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, may be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., 53010 protein or RNA) for specifictumor markers in a subject, a drug or course of treatment may beselected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in 53010 DNA may correlate 53010 drugresponse. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

Pharmaceutical Compositions

The nucleic acid and polypeptides, fragments thereof, as well asanti-53010 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositions. Suchcompositions typically include the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody can include a single treatmentor, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.

Accordingly, lower dosages and less frequent administration is oftenpossible.

Modifications such as lipidation can be used to stabilize antibodies andto enhance uptake and tissue penetration (e.g., into the brain). Amethod for lipidation of antibodies is described by Cruikshank et al.((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology14:193).

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e.,. including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive ion. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No.5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545)and analogs or homologs thereof. Therapeutic agents include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine, vinblastine, taxol andmaytansinoids). Radioactive ions include, but are not limited to iodine,yttrium and praseodymium.

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted 53010expression or activity. As used herein, the term “treatment” is definedas the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the 53010 molecules ofthe present invention or 53010 modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwanted53010 expression or activity, by administering to the subject a 53010 oran agent which modulates 53010 expression or at least one 53010activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted 53010 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 53010 aberrance, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of53010 aberrance, for example, a 53010, 53010 agonist or 53010 antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

It is possible that some 53010 disorders can be caused, at least inpart, by an abnormal level of gene product, or by the presence of a geneproduct exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of disorder symptoms.

The 53010 molecules can act as novel diagnostic targets and therapeuticagents for controlling one or more of cellular proliferative and/ordifferentiative disorders, disorders associated with bone metabolism,immune disorders, cardiovascular disorders, liver disorders, viraldiseases, blood clotting disorders, or hematopoietic disorders.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver origin.

As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung, breast, thyroid, lymphoid,gastrointestinal, and genito-urinary tract, as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Aberrant expression and/or activity of 53010 molecules may mediatedisorders associated with bone metabolism. “Bone metabolism” refers todirect or indirect effects in the formation or degeneration of bonestructures, e.g., bone formation, bone resorption, etc., which mayultimately affect the concentrations in serum of calcium and phosphate.This term also includes activities mediated by 53010 molecules effectsin bone cells, e.g. osteoclasts and osteoblasts, that may in turn resultin bone formation and degeneration. For example, 53010 molecules maysupport different activities of bone resorbing osteoclasts such as thestimulation of differentiation of monocytes and mononuclear phagocytesinto osteoclasts. Accordingly, 53010 molecules that modulate theproduction of bone cells can influence bone formation and degeneration,and thus may be used to treat bone disorders. Examples of such disordersinclude, but are not limited to, osteoporosis, osteodystrophy,osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy,osteosclerosis, anti-convulsant treatment, osteopenia,fibrogenesis-imperfecta ossium, secondary hyperparathyrodism,hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructivejaundice, drug induced metabolism, medullary carcinoma, chronic renaldisease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorptionsyndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milkfever.

The 53010 nucleic acid and protein of the invention can be used to treatand/or diagnose a variety of immune disorders. Examples of immunedisorders or diseases include, but are not limited to, autoimmunediseases (including, for example, diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversalreactions, erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis), graft-versus-host disease, cases oftransplantation, and allergy such as, atopic allergy.

Examples of disorders involving the heart or “cardiovascular disorder”include, but are not limited to, a disease, disorder, or state involvingthe cardiovascular system, e.g., the heart, the blood vessels, and/orthe blood. A cardiovascular disorder can be caused by an imbalance inarterial pressure, a malfunction of the heart, or an occlusion of ablood vessel, e.g., by a thrombus. Examples of such disorders includehypertension, atherosclerosis, coronary artery spasm, congestive heartfailure, coronary artery disease, valvular disease, arrhythmias, andcardiomyopathies.

Disorders which may be treated or diagnosed by methods described hereininclude, but are not limited to, disorders associated with anaccumulation in the liver of fibrous tissue, such as that resulting froman imbalance between production and degradation of the extracellularmatrix accompanied by the collapse and condensation of preexistingfibers. The methods described herein can be used to diagnose or treathepatocellular necrosis or injury induced by a wide variety of agentsincluding processes which disturb homeostasis, such as an inflammatoryprocess, tissue damage resulting from toxic injury or altered hepaticblood flow, and infections (e.g., bacterial, viral and parasitic). Forexample, the methods can be used for the early detection of hepaticinjury, such as portal hypertension or hepatic fibrosis. In addition,the methods can be employed to detect liver fibrosis attributed toinborn errors of metabolism, for example, fibrosis resulting from astorage disorder such as Gaucher's disease (lipid abnormalities) or aglycogen storage disease, A1-antitrypsin deficiency; a disordermediating the accumulation (e.g., storage) of an exogenous substance,for example, hemochromatosis (iron-overload syndrome) and copper storagediseases (Wilson's disease), disorders resulting in the accumulation ofa toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) andperoxisomal disorders (e.g., Zellweger syndrome). Additionally, themethods described herein may be useful for the early detection andtreatment of liver injury associated with the administration of variouschemicals or drugs, such as for example, methotrexate, isonizaid,oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, orwhich represents a hepatic manifestation of a vascular disorder such asobstruction of either the intrahepatic or extrahepatic bile flow or analteration in hepatic circulation resulting, for example, from chronicheart failure, veno-occlusive disease, portal vein thrombosis orBudd-Chiari syndrome.

Additionally, 53010 molecules may play an important role in the etiologyof certain viral diseases, including but not limited to Hepatitis B,Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 53010 activitycould be used to control viral diseases. The modulators can be used inthe treatment and/or diagnosis of viral infected tissue orvirus-associated tissue fibrosis, especially liver and liver fibrosis.Also, 53010 modulators can be used in the treatment and/or diagnosis ofvirus-associated carcinoma, especially hepatocellular cancer.

Examples of blood clotting disorders include vascular disorders causedby either partial or total occlusion of a blood vessel by a blood clot,such as stroke, deep vein thrombosis, peripheral arterial occlusion,pulmonary embolism, and myocardial thrombosis, as well as disordersinvolving defective-coagulation, e.g., hemophilia (e.g., Hemophilia Aand Hemophilia B).

As discussed, successful treatment of 53010 disorders can be broughtabout by techniques that serve to inhibit the expression or activity oftarget gene products. For example, compounds, e.g., an agent identifiedusing an assays described above, that proves to exhibit negativemodulatory activity, can be used in accordance with the invention toprevent and/or ameliorate symptoms of 53010 disorders. Such moleculescan include, but are not limited to peptides, phosphopeptides, smallorganic or inorganic molecules, or antibodies (including, for example,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or singlechain antibodies, and Fab, F(ab′)₂ and Fab expression library fragments,scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of thetarget gene can also be used in accordance with the invention to reducethe level of target gene expression, thus effectively reducing the levelof target gene activity. Still further, triple helix molecules can beutilized in reducing the level of target gene activity. Antisense,ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helixmolecules to reduce or inhibit mutant gene expression can also reduce orinhibit the transcription (triple helix) and/or translation (antisense,ribozyme) of mRNA produced by normal target gene alleles, such that theconcentration of normal target gene product present can be lower than isnecessary for a normal phenotype. In such cases, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethod. Alternatively, in instances in that the target gene encodes anextracellular protein, it can be preferable to co-administer normaltarget gene protein into the cell or tissue in order to maintain therequisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized intreating or preventing a disease characterized by 53010 expression isthrough the use of aptamer molecules specific for 53010 protein.Aptamers are nucleic acid molecules having a tertiary structure whichpermits them to specifically bind to protein ligands (see, e.g.,Osborne, et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J.(1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may inmany cases be more conveniently introduced into target cells thantherapeutic protein molecules may be, aptamers offer a method by which53010 protein activity may be specifically decreased without theintroduction of drugs or other molecules which may have pluripotenteffects.

Antibodies can be generated that are both specific for target geneproduct and that reduce target gene product activity. Such antibodiesmay, therefore, by administered in instances whereby negative modulatorytechniques are appropriate for the treatment of 53010 disorders. For adescription of antibodies, see the Antibody section above.

In circumstances wherein injection of an animal or a human subject witha 53010 protein or epitope for stimulating antibody production isharmful to the subject, it is possible to generate an immune responseagainst 53010 through the use of anti-idiotypic antibodies (see, forexample, Herlyn, D. (1999) Ann Med 31:66-78; andBhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer Treat Res.94:51-68). If an anti-idiotypic antibody is introduced into a mammal orhuman subject, it should stimulate the production of anti-anti-idiotypicantibodies, which should be specific to the 53010 protein. Vaccinesdirected to a disease characterized by 53010 expression may also begenerated in this fashion.

In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies may be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat or ameliorate 53010 disorders. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disorders.Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures as described above.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound which is able to modulate 53010 activity isused as a template, or “imprinting molecule”, to spatially organizepolymerizable monomers prior to their polymerization with catalyticreagents. The subsequent removal of the imprinted molecule leaves apolymer matrix which contains a repeated “negative image” of thecompound and is able to selectively rebind the molecule under biologicalassay conditions. A detailed review of this technique can be seen inAnsell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 andin Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such“imprinted” affinity matrixes are amenable to ligand-binding assays,whereby the immobilized monoclonal antibody component is replaced by anappropriately imprinted matrix. An example of the use of such matrixesin this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647.Through the use of isotope-labeling, the “free” concentration ofcompound which modulates the expression or activity of 53010 can bereadily monitored and used in calculations of IC₅₀. Such “imprinted”affinity matrixes can also be designed to include fluorescent groupswhose photon-emitting properties measurably change upon local andselective binding of target compound. These changes can be readilyassayed in real time using appropriate fiberoptic devices, in turnallowing the dose in a test subject to be quickly optimized based on itsindividual IC₅₀. An rudimentary example of such a “biosensor” isdiscussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating 53010expression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a 53010 or agent that modulates one or more ofthe activities of 53010 protein activity associated with the cell. Anagent that modulates 53010 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a 53010 protein (e.g., a 53010 substrate orreceptor), a 53010 antibody, a 53010 agonist or antagonist, apeptidomimetic of a 53010 agonist or antagonist, or other smallmolecule.

In one embodiment, the agent stimulates one or 53010 activities.Examples of such stimulatory agents include active 53010 protein and anucleic acid molecule encoding 53010. In another embodiment, the agentinhibits one or more 53010 activities. Examples of such inhibitoryagents include antisense 53010 nucleic acid molecules, anti-53010antibodies, and 53010 inhibitors. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a 53010 protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g., upregulates or down regulates) 53010 expression or activity. In anotherembodiment, the method involves administering a 53010 protein or nucleicacid molecule as therapy to compensate for reduced, aberrant, orunwanted 53010 expression or activity.

Stimulation of 53010 activity is desirable in situations in which 53010is abnormally downregulated and/or in which increased 53010 activity islikely to have a beneficial effect. For example, stimulation of 53010activity is desirable in situations in which a 53010 is downregulatedand/or in which increased 53010 activity is likely to have a beneficialeffect. Likewise, inhibition of 53010 activity is desirable insituations in which 53010 is abnormally upregulated and/or in whichdecreased 53010 activity is likely to have a beneficial effect.

Pharmacogenomics

The 53010 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on 53010activity (e.g., 53010 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) 53010 associated disorders (e.g.,pain or a pain-related disorder) associated with aberrant or unwanted53010 activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer a53010 molecule or 53010 modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a 53010 molecule or 53010modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. etal. (1997) Clin. Chem. 43:254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach,” can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., a 53010protein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

Alternatively, a method termed the “gene expression profiling,” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a 53010 moleculeor 53010 modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a 53010 molecule or53010 modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 53010 genes of the present invention, wherein theseproducts may be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the 53010genes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., human cells,will become sensitive to treatment with an agent that the unmodifiedtarget cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a 53010 protein can be applied in clinical trials. Forexample, the effectiveness of an agent determined by a screening assayas described herein to increase 53010 gene expression, protein levels,or upregulate 53010 activity, can be monitored in clinical trials ofsubjects exhibiting decreased 53010 gene expression, protein levels, ordownregulated 53010 activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease 53010 gene expression,protein levels, or downregulate 53010 activity, can be monitored inclinical trials of subjects exhibiting increased 53010 gene expression,protein levels, or upregulated 53010 activity. In such clinical trials,the expression or activity of a 53010 gene, and preferably, other genesthat have been implicated in, for example, a 53010-associated disordercan be used as a “read out” or markers of the phenotype of a particularcell.

53010 Informatics

The sequence of a 53010 molecule is provided in a variety of media tofacilitate use thereof. A sequence can be provided as a manufacture,other than an isolated nucleic acid or amino acid molecule, whichcontains a 53010. Such a manufacture can provide a nucleotide or aminoacid sequence, e.g., an open reading frame, in a form which allowsexamination of the manufacture using means not directly applicable toexamining the nucleotide or amino acid sequences, or a subset thereof,as they exists in nature or in purified form. The sequence informationcan include, but is not limited to, 53010 full-length nucleotide and/oramino acid sequences, partial nucleotide and/or amino acid sequences,polymorphic sequences including single nucleotide polymorphisms (SNPs),epitope sequence, and the like. In a preferred embodiment, themanufacture is a machine-readable medium, e.g., a magnetic, optical,chemical or mechanical information storage device.

As used herein, “machine-readable media” refers to any medium that canbe read and accessed directly by a machine, e.g., a digital computer oranalogue computer. Non-limiting examples of a computer include a desktopPC, laptop, mainframe, server (e.g., a web server, network server, orserver farm), handheld digital assistant, pager, mobile telephone, andthe like. The computer can be stand-alone or connected to acommunications network, e.g., a local area network (such as a VPN orintranet), a wide area network (e.g., an Extranet or the Internet), or atelephone network (e.g., a wireless, DSL, or ISDN network).Machine-readable media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as CD-ROM; electrical storage mediasuch as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybridsof these categories such as magnetic/optical storage media.

A variety of data storage structures are available to a skilled artisanfor creating a machine-readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

In a preferred embodiment, the sequence information is stored in arelational database (such as Sybase or Oracle). The database can have afirst table for storing sequence (nucleic acid and/or amino acidsequence) information. The sequence information can be stored in onefield (e.g., a first column) of a table row and an identifier for thesequence can be store in another field (e.g., a second column) of thetable row. The database can have a second table, e.g., storingannotations. The second table can have a field for the sequenceidentifier, a field for a descriptor or annotation text (e.g., thedescriptor can refer to a functionality of the sequence, a field for theinitial position in the sequence to which the annotation refers, and afield for the ultimate position in the sequence to which the annotationrefers. Non-limiting examples for annotation to nucleic acid sequencesinclude polymorphisms (e.g., SNP's) translational regulatory sites andsplice junctions. Non-limiting examples for annotations to amino acidsequence include polypeptide domains, e.g., a domain described herein;active sites and other functional amino acids; and modification sites.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. A search is used to identify fragments or regions ofthe sequences of the invention which match a particular target sequenceor target motif. The search can be a BLAST search or other routinesequence comparison, e.g., a search described herein.

Thus, in one aspect, the invention features a method of analyzing 53010,e.g., analyzing structure, function, or relatedness to one or more othernucleic acid or amino acid sequences. The method includes: providing a53010 nucleic acid or amino acid sequence; comparing the 53010 sequencewith a second sequence, e.g., one or more preferably a plurality ofsequences from a collection of sequences, e.g., a nucleic acid orprotein sequence database to thereby analyze 53010. The method can beperformed in a machine, e.g., a computer, or manually by a skilledartisan.

The method can include evaluating the sequence identity between a 53010sequence and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the Internet.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. Typical sequence lengths of a targetsequence are from about 10 to 100 amino acids or from about 30 to 300nucleotide residues. However, it is well recognized that commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but are not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBI).

Thus, the invention features a method of making a computer readablerecord of a sequence of a 53010 sequence which includes recording thesequence on a computer readable matrix. In a preferred embodiment therecord includes one or more of the following: identification of an ORF;identification of a domain, region, or site; identification of the startof transcription; identification of the transcription terminator; thefull length amino acid sequence of the protein, or a mature formthereof; the 5′ end of the translated region.

In another aspect, the invention features, a method of analyzing asequence. The method includes: providing a 53010 sequence, or record, inmachine-readable form; comparing a second sequence to the 53010sequence; thereby analyzing a sequence. Comparison can include comparingto sequences for sequence identity or determining if one sequence isincluded within the other, e.g., determining if the 53010 sequenceincludes a sequence being compared. In a preferred embodiment the 53010or second sequence is stored on a first computer, e.g., at a first siteand the comparison is performed, read, or recorded on a second computer,e.g., at a second site. E.g., the 53010 or second sequence can be storedin a public or proprietary database in one computer, and the results ofthe comparison performed, read, or recorded on a second computer. In apreferred embodiment the record includes one or more of the following:identification of an ORF; identification of a domain, region, or site;identification of the start of transcription; identification of thetranscription terminator; the full length amino acid sequence of theprotein, or a mature form thereof; the 5′ end of the translated region.

In another aspect, the invention provides a machine-readable medium forholding instructions for performing a method for determining whether asubject has a 53010-associated disease or disorder or a pre-dispositionto a 53010-associated disease or disorder, wherein the method comprisesthe steps of determining 53010 sequence information associated with thesubject and based on the 53010 sequence information, determining whetherthe subject has a 53010-associated disease or disorder or apre-disposition to a 53010-associated disease or disorder and/orrecommending a particular treatment for the disease, disorder orpre-disease condition.

The invention further provides in an electronic system and/or in anetwork, a method for determining whether a subject has a53010-associated disease or disorder or a pre-disposition to a diseaseassociated with a 53010 wherein the method comprises the steps ofdetermining 53010 sequence information associated with the subject, andbased on the 53010 sequence information, determining whether the subjecthas a 53010-associated disease or disorder or a pre-disposition to a53010-associated disease or disorder, and/or recommending a particulartreatment for the disease, disorder or pre-disease condition. In apreferred embodiment, the method further includes the step of receivinginformation, e.g., phenotypic or genotypic information, associated withthe subject and/or acquiring from a network phenotypic informationassociated with the subject. The information can be stored in adatabase, e.g., a relational database. In another embodiment, the methodfurther includes accessing the database, e.g., for records relating toother subjects, comparing the 53010 sequence of the subject to the 53010sequences in the database to thereby determine whether the subject as a53010-associated disease or disorder, or a pre-disposition for such.

The present invention also provides in a network, a method fordetermining whether a subject has a 53010 associated disease or disorderor a pre-disposition to a 53010-associated disease or disorderassociated with 53010, said method comprising the steps of receiving53010 sequence information from the subject and/or information relatedthereto, receiving phenotypic information associated with the subject,acquiring information from the network corresponding to 53010 and/orcorresponding to a 53010-associated disease or disorder (e.g., pain or apain-related disorder), and based on one or more of the phenotypicinformation, the 53010 information (e.g., sequence information and/orinformation related thereto), and the acquired information, determiningwhether the subject has a 53010-associated disease or disorder or apre-disposition to a 53010-associated disease or disorder. The methodmay further comprise the step of recommending a particular treatment forthe disease, disorder or pre-disease condition.

The present invention also provides a method for determining whether asubject has a 53010-associated disease or disorder or a pre-dispositionto a 53010-associated disease or disorder, said method comprising thesteps of receiving information related to 53010 (e.g., sequenceinformation and/or information related thereto), receiving phenotypicinformation associated with the subject, acquiring information from thenetwork related to 53010 and/or related to a 53010-associated disease ordisorder, and based on one or more of the phenotypic information, the53010 information, and the acquired information, determining whether thesubject has a 53010-associated disease or disorder or a pre-dispositionto a 53010-associated disease or disorder. The method may furthercomprise the step of recommending a particular treatment for thedisease, disorder or pre-disease condition.

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human 53010cDNA

The human 53010 nucleic acid sequence is recited as follows:

CCACGCGTCCGAAAAACAGGCCTGGAGAGCAATGTGGAGTAAGCAATGTAA (SEQ ID NO:1).TAAAAACGATTTAAAAATTATTCTTAATAAAAGTACGAATCCCA ATG CCACAGGGACTTACTTCATCTGCTTCACAATGGTGCTTTTTCCTGATTCTCCAGCCCCTGTTGGGACACAGACAGTGGGGAAAAACTGGGCCTTCTGCTGAAGGGCCACAGAGGAACACCAGGCTGGGATGGATTCAGGGCAAGCAAGTCACTGTGCTGGGAAGCCCTGTGCCTGTGAACGTGTTCCTCGGAGTCCCCTTTGCTGCTCCCCCGCTGGGATCCCTGCGATTTACGAACCCGCAGCCTGCATCGCCCTGGGATAACTTGCGAGAAGCCACCTCCTACCCTAATTTGTGCCTCCAGAACTCAGAGTGGCTGCTCTTAGATCAACACATGCTCAAGGTGCATTACCCGAAATTCGGAGTGTCAGAAGACTGCCTCTACCTGAACATCTATGCGCCTGCCCACGCCGATACAGGCTCCAAGCTCCCCGTCTTGGTGTGGTTCCCAGGAGGTGCCTTCAAGACTGGCTCAGCCTCCATCTTTGATGGGTCCGCCCTGGCTGCCTATGAGGACGTGCTGGTTGTGGTCGTCCAGTACCGGCTAGGAATATTTGGTTTCTTCACCACATGGGATCAGCATGCTCCGGGGAACTGGGCCTTCAAGGACCAGGTGGCTGCTCTGTCCTGGGTCCAGAAGAACATCGAGTTCTTCGGTGGGGACCCCAGCTCTGTGACCATCTTTGGCGAGTCCGCGGGAGCCATAAGTGTTTCTAGTCTTATACTGTCTCCCATGGCCAAAGGCTTATTCCACAAAGCCATCATGGAGAGTGGGGTGGCCATCATCCCTTACCTGGAGGCCCATGATTATGAGAAGAGTGAGGACCTGCAGGTGGTTGCACATTTCTGTGGTAACAATGCGTCAGACTCTGAGGCCCTGCTGAGGTGCCTGAGGACAAAACCCTCCAAGGAGCTGCTGACCCTCAGCCAGAAAACAAAGTCTTTCACTCGAGTGGTTGATGGTGCTTTCTTTCCTAATGAGCCTCTAGATCTATTGTCTCAGAAAGCATTTAAAGCAATTCCTTCCATCATCGGAGTCAATAACCACGAGTGTGGCTTCCTGCTGCCTATGAAGGAGGCTCCTGAGATCCTCAGTGGCTCCAACAAGTCCCTTGCCCTCCATCTGATACAAAACATCCTGCACATCCCGCCTCAGTATTTGCACCTTGTGGCTAATGAATACTTCCATGACAAGCACTCCCTGACTGAAATCCGAGACAGTCTTCTGGACTTGCTTGGAGATGTGTTCTTTGTGGTCCCTGCACTGATCACAGCTCGATATCACAGAGATGCTGGTGCACCTGTCTACTTCTATGAGTTTCGGCACCGGCCTCAGTGCTTTGAAGACACGAAGCCGGCTTTTGTCAAAGCCGACCACGCTGATGAAGTCCGCTTTGTGTTCGGTGGTGCCTTCCTGAAGGGGGACATTGTTATGTTCGAAGGAGCCACGGAGGAGGAGAAGTTACTGAGCCGGAAGATGATGAAATACTGGGCTACCTTTGCTCGAACCGGGAATCCTAATGGGAACGACCTGTCTCTGTGGCCAGCTTATAATCTGACTGAGCAGTACCTCCAGCTGGACTTGAACATGAGCCTCGGACAGAGACTCAAAGAACCGCGGGTGGATTTTTGGACCAGCACCATCCCCCTGATCCTGTCTGCCTCCGACATGCTCCACAGTCCTCTTTCTTCCTTAACTTTCCTCTCTCTCCTCCAGCCTTTCTTTTTCTTTTGTGCTCCT TGAGAAGTTATCTTTCTGTGATTTTGGTTTCCCTTCTCCTCCCATAATTTCTCCCGCAATCATTAGCTTCTTTCTGAGCTCAGCTGCTTTCTATGGGGATCCTTGCAAAACAAGCTGCTTTCGCTGATATTTTATGGACTTAGGAATGATCCTTACAGAATTCTTTTCAACATCAAAAAGTGCAATTTGTCTTGGAAGGCAACAAGATTTCTTCAATAAATTTGGAAGAGGGCTGGCCTATTAGTTGTCATAATAATGGTTTTGTAACTCATATGAAATAAAATCAGAATGTAAAATAGGAAAAAAAAAAAAAAAAA AAA.

The human 53010 sequence (SEQ ID NO: 1) is approximately 2158nucleotides long. The nucleic acid sequence includes an initiation codon(ATG) and a termination codon (TGA) which are underscored above. Theregion between and inclusive of the initiation codon and the terminationcodon is a methionine-initiated coding sequence of about 1746nucleotides, including the termination codon (nucleotides indicated as“coding” of SEQ ID NO:1; SEQ ID NO:3). The coding sequence encodes a 581amino acid protein (SEQ ID NO:2), which is recited as follows:

MPQGLTSSASQWCFFLILQPLLGHRQWGKTGPSAEGPQRNTRLGWIQGKQVTVL (SEQ ID NO:2).GSPVPVNVFLGVPFAAPPLGSLRFTNPQPASPWDNLREATSYPNLCLQNSEWLLLDQHMLKVHYPKFGVSEDCLYLNIYAPAHADTGSKLPVLVWFPGGAFKTGSASIFDGSALAAYEDVLVVVVQYRLGIFGFFTTWDQHAPGNWAFKDQVAALSWVQKNIEFFGGDPSSVTIFGESAGAISVSSLILSPMAKGLFHKAIMESGVAIIPYLEAHDYEKSEDLQVVAHFCGNNASDSEALLRCLRTKPSKELLTLSQKTKSFTRVVDGAFFPNEPLDLLSQKAFKAIPSIIGVNNHECGFLLPMKEAPEILSGSNKSLALHLIQNILHIPPQYLHLVANEYFHDKHSLTEIRDSLLDLLGDVFFVVPALITARYHRDAGAPVYFYEFRHRPQCFEDTKPAFVKADHADEVRFVFGGAFLKGDIVMFEGATEEEKLLSRKMMKYWATFARTGNPNGNDLSLWPAYNLTEQYLQLDLNMSLGQRLKEPRVDFWTSTIPLILSASDMLHSPLSSLTFLSLLQPFFFFCAP.

Example 2 Tissue Distribution of 53010 mRNA by TaqMan Analysis

Endogenous human 53010 gene expression was determined using thePerkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMantechnology. Briefly, TaqMan technology relies on standard RT-PCR withthe addition of a third gene-specific oligonucleotide (referred to as aprobe) which has a fluorescent dye coupled to its 5′ end (typically6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When thefluorescently tagged oligonucleotide is intact, the fluorescent signalfrom the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolyticactivity of Taq polymerase digests the labeled primer, producing a freenucleotide labeled with 6-FAM, which is now detected as a fluorescentsignal. The PCR cycle where fluorescence is first released and detectedis directly proportional to the starting amount of the gene of interestin the test sample, thus providing a quantitative measure of the initialtemplate concentration. Samples can be internally controlled by theaddition of a second set of primers/probe specific for a housekeepinggene such as GAPDH which has been labeled with a different fluorophoreon the 5′ end (typically VIC).

To determine the level of 53010 in various human tissues a primer/probeset was designed. Total RNA was prepared from a series of human tissuesusing an RNeasy kit from Qiagen. First strand cDNA was prepared from 1μg total RNA using an oligo-dT primer and Superscript II reversetranscriptase (Gibco/BRL). cDNA obtained from approximately 50 ng totalRNA was used per TaqMan reaction. Tissues tested include the humantissues and several cell lines shown in Tables 1-3. As shown in Table 1,53010 mRNA was detected in brain, spinal cord, dorsal root ganglion(DRG), and kidney. As shown in Table 2, 53010 mRNA was detected intestes, brain, spinal cord, dorsal root ganglion (DRG), skin, and liver.As shown in Table 3, 53010 mRNA was detected in brain, spinal cord,dorsal root ganglion (DRG), and superior cervical ganglion (SCG).

TABLE 1 Expression of 53010 Tissue Type Relative Expression Arterynormal 0 Aorta diseased 0 Vein normal 0 Coronary Smooth Muscle Cells 0Human Umbilical Vein Endothelial Cells 0 Hemangioma 0 Heart normal 0Heart congestive heart failure 0 Kidney 1.4298 Skeletal Muscle 0 Adiposenormal 0 Pancreas 0 primary osteoblasts 0 Osteoclasts (diff) 0 Skinnormal 0 Spinal cord normal 0.9143 Brain Cortex normal 16.4588 BrainHypothalamus normal 0 Nerve 0 Dorsal Root Ganglion 1.1102 Breast normal0 Breast tumor 0 Ovary normal 0 Ovary Tumor 0 Prostate Normal 0 ProstateTumor 0 Salivary glands 0 Colon normal 0 Colon Tumor 0 Lung normal 0Lung tumor 0 Lung Chronic Obstructive Pulmonary Disease 0 ColonInflammatory Bowel Disease 0 Liver normal 0 Liver fibrosis 0 Spleennormal 0 Tonsil normal 0.0466 Lymph node normal 0 Small intestine normal0 Macrophages 0 Synovium 0 Bone Marrow, Mononuclear Cells 0 Activatedperipheral blood mononuclear cells 0 Neutrophils 0 Megakaryocytes 0Erythroid 0 positive control 18.5171

TABLE 2 Expression of 53010 in Human Tissues Tissue Type RelativeExpression Adrenal Gland 0.02 Brain 0.86 Heart 0.00 Kidney 0.01 Liver0.14 Lung 0.00 Mammary Gland 0.00 Pancreas 0.01 Placenta 0.00 Prostate0.01 Salivary Gland 0.01 Muscle 0.01 Small Intestine 0.00 Spleen 0.00Stomach 0.01 Testes 3.30 Thymus 0.02 Trachea 0.06 Uterus 0.00 SpinalCord 0.25 DRG 0.07 Skin 0.22

TABLE 3 Expression of 53010 in Rat Tissues Tissue Relative ExpressionBrain 0.006 Spinal Cord 0.012 Dorsal Root Ganglia 0.008 SuperiorCervical Ganglion 0.002 Hairy Skin 0.000 Gastro Muscle 0.000 Heart 0.000Kidney 0.000 Liver 0.000 Lung 0.000 Spleen 0.000 Aorta 0.000 AdrenalGland 0.000 Salivary Gland 0.000 Thyroid 0.000 Prostate 0.000 Thymus0.000 Trachea 0.000 Esophagus 0.000 Duodenum 0.000 Diaphragm 0.000

The regulation of the rat orthologue of the 53010 gene was evaluated inrodent models of pain response. In these experiments, 53010 expressionwas evaluated at various days (D) following the treatment. Table 4 showsthe regulation of 53010 expression in dorsal root ganglion (DRG)following CFA injection (days 1, 3, 7, 14, and 28), axotomy (AX; days 1,3, 7, 14, and 28), and CCI (days 3, 7, 10, 14, and 28). Table 5 showsthe regulation of 53010 expression in the spinal cord (SC) following CFAinjection (days 3, 7, 14, and 28), axotomy (AX; days 1, 3, 7, 14, and28), and CCI (days 3, 7, and 14).

TABLE 4 Regulation of 53010 Expression in Dorsal Root Ganglion SampleRelative Expression Naïve DRG 0.00562389 CCI D3, ipsilateral, DRG0.00349809 CCI D7, ipsilateral, DRG 0.00397669 CCI D10, ipsilateral, DRG0.00421803 CCI D14, ipsilateral, DRG 0.00280222 CCI D28, ipsilateral,DRG 0.00228401 Naïve DRG 0.00562389 CFA D1 ipsilateral, DRG 0.00192062CFA D3 ipsilateral, DRG 0.00408849 CFA D7 ipsilateral, DRG 0.00226039CFA D14 ipsilateral, DRG 0.00227611 CFA D28 ipsilateral, DRG 0.00588307Naïve DRG 0.00562389 AX D1, ipsilateral, DRG 0.00267877 AX D3,ipsilateral, DRG 0.00173696 AX D7, ipsilateral, DRG 0.00266026 AX D14,ipsilateral, DRG 0.00118228 AX D28, ipsilateral, DRG 0.00421803

TABLE 5 Regulation of 53010 Expression in Spinal Cord Sample RelativeExpression Naïve SC 0.00394922 CCI D3, ipsilateral, SC 0.00438194 CCID7, ipsilateral, SC 0.00496423 CCI D14, ipsilateral, SC 0.01109293 NaïveSC 0.00394922 CFA D3 ipsilateral, SC 0.00400435 CFA D7 ipsilateral, SC0.00843606 CFA D14 ipsilateral, SC 0.00162627 CFA D28 ipsilateral, SC0.00530212 Naïve SC 0.00739509 AX D1, ipsilateral, SC 0.00721785 AX D3,ipsilateral, SC 0.006876 AX D7, ipsilateral, SC 0.00843606 AX D14,ipsilateral, SC 0.00426211 AX D28, ipsilateral, SC 0.00359644

Example 3 Tissue Distribution of 53010 mRNA by Northern Analysis

Northern blot hybridizations with various RNA samples can be performedunder standard conditions and washed under stringent conditions, i.e.,0.2× SSC at 65° C. A DNA probe corresponding to all or a portion of the53010 cDNA (SEQ ID NO:1) can be used. The DNA was radioactively labeledwith ³²P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.)according to the instructions of the supplier. Filters containing mRNAfrom mouse hematopoietic and endocrine tissues, and cancer cell lines(Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridizationsolution (Clontech) and washed at high stringency according tomanufacturer's recommendations.

Example 4 Recombinant Expression of 53010 in Bacterial Cells

In this example, 53010 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 53010 isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-53010 fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 5 Expression of Recombinant 53010 Protein in COS Cells

To express the 53010 gene in COS cells (e.g., COS-7 cells, CV-1 originSV40 cells; Gluzman (1981) CellI23:175-182), the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 53010 protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the 53010 DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 53010coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 53010 coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the 53010 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the 53010-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual. 2nd, ed. Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. The expression of the 53010 polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) using an HA specificmonoclonal antibody. Briefly, the cells are labeled for 8 hours with³⁵S-methionine (or ³⁵S-cysteine). The culture media are then collectedand the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1%NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate andthe culture media are precipitated with an HA specific monoclonalantibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 53010 coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the 53010polypeptide is detected by radiolabelling and immunoprecipitation usinga 53010 specific monoclonal antibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

6 1 2158 DNA Homo sapiens CDS (96)...(1838) 1 ccacgcgtcc gaaaaacaggcctggagagc aatgtggagt aagcaatgta ataaaaacga 60 tttaaaaatt attcttaataaaagtacgaa tccca atg cca cag gga ctt act 113 Met Pro Gln Gly Leu Thr 1 5tca tct gct tca caa tgg tgc ttt ttc ctg att ctc cag ccc ctg ttg 161 SerSer Ala Ser Gln Trp Cys Phe Phe Leu Ile Leu Gln Pro Leu Leu 10 15 20 ggacac aga cag tgg gga aaa act ggg cct tct gct gaa ggg cca cag 209 Gly HisArg Gln Trp Gly Lys Thr Gly Pro Ser Ala Glu Gly Pro Gln 25 30 35 agg aacacc agg ctg gga tgg att cag ggc aag caa gtc act gtg ctg 257 Arg Asn ThrArg Leu Gly Trp Ile Gln Gly Lys Gln Val Thr Val Leu 40 45 50 gga agc cctgtg cct gtg aac gtg ttc ctc gga gtc ccc ttt gct gct 305 Gly Ser Pro ValPro Val Asn Val Phe Leu Gly Val Pro Phe Ala Ala 55 60 65 70 ccc ccg ctggga tcc ctg cga ttt acg aac ccg cag cct gca tcg ccc 353 Pro Pro Leu GlySer Leu Arg Phe Thr Asn Pro Gln Pro Ala Ser Pro 75 80 85 tgg gat aac ttgcga gaa gcc acc tcc tac cct aat ttg tgc ctc cag 401 Trp Asp Asn Leu ArgGlu Ala Thr Ser Tyr Pro Asn Leu Cys Leu Gln 90 95 100 aac tca gag tggctg ctc tta gat caa cac atg ctc aag gtg cat tac 449 Asn Ser Glu Trp LeuLeu Leu Asp Gln His Met Leu Lys Val His Tyr 105 110 115 ccg aaa ttc ggagtg tca gaa gac tgc ctc tac ctg aac atc tat gcg 497 Pro Lys Phe Gly ValSer Glu Asp Cys Leu Tyr Leu Asn Ile Tyr Ala 120 125 130 cct gcc cac gccgat aca ggc tcc aag ctc ccc gtc ttg gtg tgg ttc 545 Pro Ala His Ala AspThr Gly Ser Lys Leu Pro Val Leu Val Trp Phe 135 140 145 150 cca gga ggtgcc ttc aag act ggc tca gcc tcc atc ttt gat ggg tcc 593 Pro Gly Gly AlaPhe Lys Thr Gly Ser Ala Ser Ile Phe Asp Gly Ser 155 160 165 gcc ctg gctgcc tat gag gac gtg ctg gtt gtg gtc gtc cag tac cgg 641 Ala Leu Ala AlaTyr Glu Asp Val Leu Val Val Val Val Gln Tyr Arg 170 175 180 cta gga atattt ggt ttc ttc acc aca tgg gat cag cat gct ccg ggg 689 Leu Gly Ile PheGly Phe Phe Thr Thr Trp Asp Gln His Ala Pro Gly 185 190 195 aac tgg gccttc aag gac cag gtg gct gct ctg tcc tgg gtc cag aag 737 Asn Trp Ala PheLys Asp Gln Val Ala Ala Leu Ser Trp Val Gln Lys 200 205 210 aac atc gagttc ttc ggt ggg gac ccc agc tct gtg acc atc ttt ggc 785 Asn Ile Glu PhePhe Gly Gly Asp Pro Ser Ser Val Thr Ile Phe Gly 215 220 225 230 gag tccgcg gga gcc ata agt gtt tct agt ctt ata ctg tct ccc atg 833 Glu Ser AlaGly Ala Ile Ser Val Ser Ser Leu Ile Leu Ser Pro Met 235 240 245 gcc aaaggc tta ttc cac aaa gcc atc atg gag agt ggg gtg gcc atc 881 Ala Lys GlyLeu Phe His Lys Ala Ile Met Glu Ser Gly Val Ala Ile 250 255 260 atc ccttac ctg gag gcc cat gat tat gag aag agt gag gac ctg cag 929 Ile Pro TyrLeu Glu Ala His Asp Tyr Glu Lys Ser Glu Asp Leu Gln 265 270 275 gtg gttgca cat ttc tgt ggt aac aat gcg tca gac tct gag gcc ctg 977 Val Val AlaHis Phe Cys Gly Asn Asn Ala Ser Asp Ser Glu Ala Leu 280 285 290 ctg aggtgc ctg agg aca aaa ccc tcc aag gag ctg ctg acc ctc agc 1025 Leu Arg CysLeu Arg Thr Lys Pro Ser Lys Glu Leu Leu Thr Leu Ser 295 300 305 310 cagaaa aca aag tct ttc act cga gtg gtt gat ggt gct ttc ttt cct 1073 Gln LysThr Lys Ser Phe Thr Arg Val Val Asp Gly Ala Phe Phe Pro 315 320 325 aatgag cct cta gat cta ttg tct cag aaa gca ttt aaa gca att cct 1121 Asn GluPro Leu Asp Leu Leu Ser Gln Lys Ala Phe Lys Ala Ile Pro 330 335 340 tccatc atc gga gtc aat aac cac gag tgt ggc ttc ctg ctg cct atg 1169 Ser IleIle Gly Val Asn Asn His Glu Cys Gly Phe Leu Leu Pro Met 345 350 355 aaggag gct cct gag atc ctc agt ggc tcc aac aag tcc ctt gcc ctc 1217 Lys GluAla Pro Glu Ile Leu Ser Gly Ser Asn Lys Ser Leu Ala Leu 360 365 370 catctg ata caa aac atc ctg cac atc ccg cct cag tat ttg cac ctt 1265 His LeuIle Gln Asn Ile Leu His Ile Pro Pro Gln Tyr Leu His Leu 375 380 385 390gtg gct aat gaa tac ttc cat gac aag cac tcc ctg act gaa atc cga 1313 ValAla Asn Glu Tyr Phe His Asp Lys His Ser Leu Thr Glu Ile Arg 395 400 405gac agt ctt ctg gac ttg ctt gga gat gtg ttc ttt gtg gtc cct gca 1361 AspSer Leu Leu Asp Leu Leu Gly Asp Val Phe Phe Val Val Pro Ala 410 415 420ctg atc aca gct cga tat cac aga gat gct ggt gca cct gtc tac ttc 1409 LeuIle Thr Ala Arg Tyr His Arg Asp Ala Gly Ala Pro Val Tyr Phe 425 430 435tat gag ttt cgg cac cgg cct cag tgc ttt gaa gac acg aag ccg gct 1457 TyrGlu Phe Arg His Arg Pro Gln Cys Phe Glu Asp Thr Lys Pro Ala 440 445 450ttt gtc aaa gcc gac cac gct gat gaa gtc cgc ttt gtg ttc ggt ggt 1505 PheVal Lys Ala Asp His Ala Asp Glu Val Arg Phe Val Phe Gly Gly 455 460 465470 gcc ttc ctg aag ggg gac att gtt atg ttc gaa gga gcc acg gag gag 1553Ala Phe Leu Lys Gly Asp Ile Val Met Phe Glu Gly Ala Thr Glu Glu 475 480485 gag aag tta ctg agc cgg aag atg atg aaa tac tgg gct acc ttt gct 1601Glu Lys Leu Leu Ser Arg Lys Met Met Lys Tyr Trp Ala Thr Phe Ala 490 495500 cga acc ggg aat cct aat ggg aac gac ctg tct ctg tgg cca gct tat 1649Arg Thr Gly Asn Pro Asn Gly Asn Asp Leu Ser Leu Trp Pro Ala Tyr 505 510515 aat ctg act gag cag tac ctc cag ctg gac ttg aac atg agc ctc gga 1697Asn Leu Thr Glu Gln Tyr Leu Gln Leu Asp Leu Asn Met Ser Leu Gly 520 525530 cag aga ctc aaa gaa ccg cgg gtg gat ttt tgg acc agc acc atc ccc 1745Gln Arg Leu Lys Glu Pro Arg Val Asp Phe Trp Thr Ser Thr Ile Pro 535 540545 550 ctg atc ctg tct gcc tcc gac atg ctc cac agt cct ctt tct tcc tta1793 Leu Ile Leu Ser Ala Ser Asp Met Leu His Ser Pro Leu Ser Ser Leu 555560 565 act ttc ctc tct ctc ctc cag cct ttc ttt ttc ttt tgt gct cct 1838Thr Phe Leu Ser Leu Leu Gln Pro Phe Phe Phe Phe Cys Ala Pro 570 575 580tgagaagtta tctttctgtg attttggttt cccttctcct cccataattt ctcccgcaat 1898cattagcttc tttctgagct cagctgcttt ctatggggat ccttgcaaaa caagctgctt 1958tcgctgatat tttatggact taggaatgat ccttacagaa ttcttttcaa catcaaaaag 2018tgcaatttgt cttggaaggc aacaagattt cttcaataaa tttggaagag ggctggccta 2078ttagttgtca taataatggt tttgtaactc atatgaaata aaatcagaat gtaaaatagg 2138aaaaaaaaaa aaaaaaaaaa 2158 2 581 PRT Homo sapiens 2 Met Pro Gln Gly LeuThr Ser Ser Ala Ser Gln Trp Cys Phe Phe Leu 1 5 10 15 Ile Leu Gln ProLeu Leu Gly His Arg Gln Trp Gly Lys Thr Gly Pro 20 25 30 Ser Ala Glu GlyPro Gln Arg Asn Thr Arg Leu Gly Trp Ile Gln Gly 35 40 45 Lys Gln Val ThrVal Leu Gly Ser Pro Val Pro Val Asn Val Phe Leu 50 55 60 Gly Val Pro PheAla Ala Pro Pro Leu Gly Ser Leu Arg Phe Thr Asn 65 70 75 80 Pro Gln ProAla Ser Pro Trp Asp Asn Leu Arg Glu Ala Thr Ser Tyr 85 90 95 Pro Asn LeuCys Leu Gln Asn Ser Glu Trp Leu Leu Leu Asp Gln His 100 105 110 Met LeuLys Val His Tyr Pro Lys Phe Gly Val Ser Glu Asp Cys Leu 115 120 125 TyrLeu Asn Ile Tyr Ala Pro Ala His Ala Asp Thr Gly Ser Lys Leu 130 135 140Pro Val Leu Val Trp Phe Pro Gly Gly Ala Phe Lys Thr Gly Ser Ala 145 150155 160 Ser Ile Phe Asp Gly Ser Ala Leu Ala Ala Tyr Glu Asp Val Leu Val165 170 175 Val Val Val Gln Tyr Arg Leu Gly Ile Phe Gly Phe Phe Thr ThrTrp 180 185 190 Asp Gln His Ala Pro Gly Asn Trp Ala Phe Lys Asp Gln ValAla Ala 195 200 205 Leu Ser Trp Val Gln Lys Asn Ile Glu Phe Phe Gly GlyAsp Pro Ser 210 215 220 Ser Val Thr Ile Phe Gly Glu Ser Ala Gly Ala IleSer Val Ser Ser 225 230 235 240 Leu Ile Leu Ser Pro Met Ala Lys Gly LeuPhe His Lys Ala Ile Met 245 250 255 Glu Ser Gly Val Ala Ile Ile Pro TyrLeu Glu Ala His Asp Tyr Glu 260 265 270 Lys Ser Glu Asp Leu Gln Val ValAla His Phe Cys Gly Asn Asn Ala 275 280 285 Ser Asp Ser Glu Ala Leu LeuArg Cys Leu Arg Thr Lys Pro Ser Lys 290 295 300 Glu Leu Leu Thr Leu SerGln Lys Thr Lys Ser Phe Thr Arg Val Val 305 310 315 320 Asp Gly Ala PhePhe Pro Asn Glu Pro Leu Asp Leu Leu Ser Gln Lys 325 330 335 Ala Phe LysAla Ile Pro Ser Ile Ile Gly Val Asn Asn His Glu Cys 340 345 350 Gly PheLeu Leu Pro Met Lys Glu Ala Pro Glu Ile Leu Ser Gly Ser 355 360 365 AsnLys Ser Leu Ala Leu His Leu Ile Gln Asn Ile Leu His Ile Pro 370 375 380Pro Gln Tyr Leu His Leu Val Ala Asn Glu Tyr Phe His Asp Lys His 385 390395 400 Ser Leu Thr Glu Ile Arg Asp Ser Leu Leu Asp Leu Leu Gly Asp Val405 410 415 Phe Phe Val Val Pro Ala Leu Ile Thr Ala Arg Tyr His Arg AspAla 420 425 430 Gly Ala Pro Val Tyr Phe Tyr Glu Phe Arg His Arg Pro GlnCys Phe 435 440 445 Glu Asp Thr Lys Pro Ala Phe Val Lys Ala Asp His AlaAsp Glu Val 450 455 460 Arg Phe Val Phe Gly Gly Ala Phe Leu Lys Gly AspIle Val Met Phe 465 470 475 480 Glu Gly Ala Thr Glu Glu Glu Lys Leu LeuSer Arg Lys Met Met Lys 485 490 495 Tyr Trp Ala Thr Phe Ala Arg Thr GlyAsn Pro Asn Gly Asn Asp Leu 500 505 510 Ser Leu Trp Pro Ala Tyr Asn LeuThr Glu Gln Tyr Leu Gln Leu Asp 515 520 525 Leu Asn Met Ser Leu Gly GlnArg Leu Lys Glu Pro Arg Val Asp Phe 530 535 540 Trp Thr Ser Thr Ile ProLeu Ile Leu Ser Ala Ser Asp Met Leu His 545 550 555 560 Ser Pro Leu SerSer Leu Thr Phe Leu Ser Leu Leu Gln Pro Phe Phe 565 570 575 Phe Phe CysAla Pro 580 3 1746 DNA Homo sapiens 3 atgccacagg gacttacttc atctgcttcacaatggtgct ttttcctgat tctccagccc 60 ctgttgggac acagacagtg gggaaaaactgggccttctg ctgaagggcc acagaggaac 120 accaggctgg gatggattca gggcaagcaagtcactgtgc tgggaagccc tgtgcctgtg 180 aacgtgttcc tcggagtccc ctttgctgctcccccgctgg gatccctgcg atttacgaac 240 ccgcagcctg catcgccctg ggataacttgcgagaagcca cctcctaccc taatttgtgc 300 ctccagaact cagagtggct gctcttagatcaacacatgc tcaaggtgca ttacccgaaa 360 ttcggagtgt cagaagactg cctctacctgaacatctatg cgcctgccca cgccgataca 420 ggctccaagc tccccgtctt ggtgtggttcccaggaggtg ccttcaagac tggctcagcc 480 tccatctttg atgggtccgc cctggctgcctatgaggacg tgctggttgt ggtcgtccag 540 taccggctag gaatatttgg tttcttcaccacatgggatc agcatgctcc ggggaactgg 600 gccttcaagg accaggtggc tgctctgtcctgggtccaga agaacatcga gttcttcggt 660 ggggacccca gctctgtgac catctttggcgagtccgcgg gagccataag tgtttctagt 720 cttatactgt ctcccatggc caaaggcttattccacaaag ccatcatgga gagtggggtg 780 gccatcatcc cttacctgga ggcccatgattatgagaaga gtgaggacct gcaggtggtt 840 gcacatttct gtggtaacaa tgcgtcagactctgaggccc tgctgaggtg cctgaggaca 900 aaaccctcca aggagctgct gaccctcagccagaaaacaa agtctttcac tcgagtggtt 960 gatggtgctt tctttcctaa tgagcctctagatctattgt ctcagaaagc atttaaagca 1020 attccttcca tcatcggagt caataaccacgagtgtggct tcctgctgcc tatgaaggag 1080 gctcctgaga tcctcagtgg ctccaacaagtcccttgccc tccatctgat acaaaacatc 1140 ctgcacatcc cgcctcagta tttgcaccttgtggctaatg aatacttcca tgacaagcac 1200 tccctgactg aaatccgaga cagtcttctggacttgcttg gagatgtgtt ctttgtggtc 1260 cctgcactga tcacagctcg atatcacagagatgctggtg cacctgtcta cttctatgag 1320 tttcggcacc ggcctcagtg ctttgaagacacgaagccgg cttttgtcaa agccgaccac 1380 gctgatgaag tccgctttgt gttcggtggtgccttcctga agggggacat tgttatgttc 1440 gaaggagcca cggaggagga gaagttactgagccggaaga tgatgaaata ctgggctacc 1500 tttgctcgaa ccgggaatcc taatgggaacgacctgtctc tgtggccagc ttataatctg 1560 actgagcagt acctccagct ggacttgaacatgagcctcg gacagagact caaagaaccg 1620 cgggtggatt tttggaccag caccatccccctgatcctgt ctgcctccga catgctccac 1680 agtcctcttt cttccttaac tttcctctctctcctccagc ctttcttttt cttttgtgct 1740 ccttga 1746 4 574 PRT ArtificialSequence Consensus sequence 4 Gly Lys Val Arg Gly Val Asn Glu Lys ThrAsp Asn Gly Glu Gln Ser 1 5 10 15 Val Tyr Ser Phe Leu Gly Ile Pro TyrAla Glu Pro Pro Val Gly Asn 20 25 30 Leu Arg Phe Lys Ala Pro Gln Pro TyrLys Glu Pro Trp Ser Asp Val 35 40 45 Leu Asp Ala Thr Lys Tyr Pro Pro SerCys Leu Gln Asp Asp Asp Phe 50 55 60 Gly Phe Ser Leu Ser Asp Leu Lys ValAla Leu Lys Met Leu Ser Leu 65 70 75 80 Gly Trp Asn Lys Leu Val Gly LeuLys Leu Ser Glu Asp Cys Leu Tyr 85 90 95 Leu Asn Val Tyr Thr Pro Lys AsnThr Lys Pro Asn Ser Lys Leu Pro 100 105 110 Val Met Val Trp Ile His GlyGly Gly Phe Met Phe Gly Ser Gly His 115 120 125 Ser Leu Pro Leu Ser LeuTyr Asp Gly Glu Ser Leu Ala Arg Glu Gly 130 135 140 Asn Val Ile Val ValSer Ile Asn Tyr Arg Leu Gly Pro Leu Gly Phe 145 150 155 160 Leu Ser ThrGly Asp Asp Lys Leu Pro Gly Ser Gly Asn Tyr Gly Leu 165 170 175 Leu AspGln Arg Leu Ala Leu Lys Trp Val Gln Asp Asn Ile Ala Ala 180 185 190 PheGly Gly Asp Pro Asn Ser Val Thr Ile Phe Gly Glu Ser Ala Gly 195 200 205Ala Ala Ser Val Ser Leu Leu Leu Leu Ser Asn Gly Gly Asp Asn Pro 210 215220 Pro Ser Ser Lys Gly Leu Phe His Arg Ala Ile Ser Gln Ser Gly Ser 225230 235 240 Ala Leu Ser Pro Trp Ala Ile Gln Ser Glu Ser Asn Ala Arg GlyArg 245 250 255 Ala Lys Glu Leu Ala Arg Leu Leu Gly Cys Asn Glu Thr SerSer Ser 260 265 270 Glu Leu Leu Asp Cys Leu Arg Ser Lys Ser Ala Glu GluLeu Leu Glu 275 280 285 Ala Thr Arg Ser Phe Leu Leu Phe Glu Tyr Val ProPhe Leu Pro Leu 290 295 300 Phe Leu Ala Phe Gly Pro Val Val Asp Gly AspAsp Ala Pro Glu Ala 305 310 315 320 Phe Ile Pro Glu Asp Pro Glu Glu LeuIle Lys Glu Gly Lys Phe Ala 325 330 335 Asp Val Pro Tyr Leu Ile Gly ValThr Lys Asp Glu Gly Gly Tyr Phe 340 345 350 Ala Ala Met Leu Leu Asn AlaSer Ser Lys Gly Glu Asp Glu Leu Lys 355 360 365 Lys Glu Thr Asn Pro AspVal Trp Leu Glu Leu Leu Lys Tyr Leu Leu 370 375 380 Phe Tyr Ala Ser GluAla Leu Asn Ile Lys Asp Met Asp Asp Leu Ala 385 390 395 400 Asp Lys ValLeu Glu Lys Tyr Pro Gly Asp Val Asp Asp Phe Ser Val 405 410 415 Glu SerArg Lys Pro Asn Leu Gln Asp Met Leu Thr Asp Leu Leu Phe 420 425 430 LysCys Pro Thr Arg Val Ala Ala Asp Leu His Ala Lys His Gly Gly 435 440 445Ser Pro Val Tyr Ala Tyr Val Phe Asp His Pro Ala Ser Phe Gly Ile 450 455460 Gly Gln Phe Leu Ala Lys Arg Val Asp Pro Glu Phe Gly Gly Ala Val 465470 475 480 His Gly Asp Glu Ile Phe Phe Val Phe Gly Asn Pro Leu Leu LysGlu 485 490 495 Gln Leu Tyr Lys Ala Thr Glu Glu Glu Glu Lys Ser Ser SerLys Thr 500 505 510 Met Met Asn Tyr Trp Ala Asn Phe Ala Lys Thr Gly AsnPro Asn Asn 515 520 525 Gly Thr Ser Asn Gly Leu Val Val Trp Pro Lys TyrThr Ser Glu Glu 530 535 540 Gln Lys Tyr Ser Leu Leu Ile Leu Leu Thr ThrIle Thr Ala Gln Lys 545 550 555 560 Leu Lys Ala Arg Asp Pro Arg Lys ValLeu Cys Asn Phe Trp 565 570 5 16 PRT Artificial Sequence Exemplary motif5 Phe Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Ser Xaa Gly 1 5 1015 6 11 PRT Artificial Sequence Exemplary motif 6 Xaa Asp Cys Leu XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: a) a nucleic acid comprising the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, or a full complement thereof; andb) a nucleic acid molecule which encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 2. The nucleic acid molecule ofclaim 1, further comprising vector nucleic acid sequences.
 3. Thenucleic acid molecule of claim 1, further comprising nucleic acidsequences encoding a heterologous polypeptide.
 4. A host cell whichcontains the nucleic acid molecule of claim
 1. 5. A method for producinga polypeptide comprising the amino acid sequence of SEQ ID NO:2, themethod comprising culturing the host cell of claim 4 under conditions inwhich the nucleic acid molecule is expressed.